It is well documented that the ventrolateral column of the midbrain periaqueductal gray (vl-PAG) plays a vital role in the modulation of spinal nociceptive neurons. It has been shown that this antinociceptive effect is expressed by the vl-PAG through the activation of spinally projecting serotonergic and noradrenergic pathways arising from the rostral ventromedial medulla (RVM) and pontine noradrenergic nuclei. However, it has recently been reported that monosynaptic excitation of RVM and pontine noradrenergic neurons is not necessary for the antinociceptive effects of the vl-PAG, thus necessitating the reevaluation of this theory. Using anatomical and electrophysiological techniques, we intended to investigate further the functional link between the vl-PAG and the RVM and pontine noradrenergic nuclei. With neuronal tracing and immunocytochemical techniques, we studied the projections of the vl-PAG to the pons and medulla oblongata. With pre- and post-embedding immunohistochemical methods we investigated the neurotransmitter profile of axon terminals that arise from the vl-PAG as well as their postsynaptic targets. In addition, with in vivo electrophysiological recording, we analysed the responses of RVM neurons evoked by electrical stimulation of the vl-PAG. Confirming previous results, we demonstrated that a small proportion of the PAG efferents terminated in the RVM and pontine noradrenergic nuclei. However, revealing serotonin and tyrosine hydroxylase immunoreactive neurons (SIR, THIR), we found that, in contrast to previous reports, PAG efferents make relatively few appositions with SIR and THIR neurons, they mostly terminate in non-SIR and non-THIR territories within the RVM and pontine noradrenergic areas. In addition, we found that the intermediate subdivision of the ponto-bulbar reticular formation (iRF) receives a substantial projection from the vl-PAG. We have also revealed that many of the terminals of the vl-PAG efferent fibres make close appositions with preproenkephalin immunoreactive neurons within the iRF. Our physiological study showed that the vl-PAG stimulation evokes excitation of RVM cells with different onset latencies. Some of the RVM neurons respond to vl-PAG stimulation with short onset latency (3.6 + 0.9 ms), while others show delayed responses, with nearly four times longer (14.8 + 3 ms) onset latency. The findings show that RVM neurons receive heterogeneous, partly monosynaptic and partly polysynaptic inputs from the vl-PAG. Our results suggest that neural activities of the vl-PAG may influence the nociceptive information processing mechanisms of the spinal cord through an intricate interneuronal circuit. Most probably signals from the vl-PAG substantially influence the excitation level of neurons in the iRF. Here the PAG signals may activate neural circuits that forward neural activities towards the spinal cord or more likely towards the RVM and pontine noradrenergic cell groups. A second group of PAG efferents terminate within the RVM and pontine noradrenergic cell groups on non-SIR and non-THIR neurons, activating interneural circuits that presumably further process the incoming signals and forwards them to spinally projecting neurons. A third group of efferent fibres may establish monosynaptic contacts with spinally projecting serotonergic and noradrenergic neurons. The spinally projecting neurons then presumably integrate the monosynaptic inputs and signals coming from the activated intra- and extranuclear interneural circuits. In case of suprathreshold activation they may conduct volleys to the spinal dorsal horn, where they inhibit spinal neural circuits underlying nociceptive information processing, resulting in analgesia and attenuation of pain behaviour.