Genetically modified T cells expressing chimeric antigen receptors (CARs) specific for tumor associated antigens (TAA) have shown to be effective in treatment of various B cell malignancies, however their safety and clinical efficacy against solid tumors remains poor. The performed experiments help us understand how currently evolving chimeric antigen receptor T cell-based therapies function at the molecular level. In our project we aimed to compare the short- and long-term activation of retrovirally transduced second generation HER specific CAR T cells expressing CD28 or 41BB costimulatory endodomains and investigate the importance of the various signal enhancer molecules. CAR T cells were generated by retroviral transduction on Retronectin coated plate. Viral particles encoded αHER2_scFv-CD28-CD3ζ or αHER2_scFv-41BB-CD3ζ CAR constructs. The optimized transduction protocol yielded cells with over 90% CD3 positivity and over 60% HER2-CAR positivity for both constructs as confirmed by flow cytometry. First, we characterized the impact of target density on short-term CAR T cell activation in cytokine release assays. We found that according to our previous results, CAR T cells expressing a CD28 costimulatory endodomain shown significantly better activation in presence of target antigen, moreover increased density of immobilized Fc conjugated recombinant HER2 protein (0,0001 μg/ml vs 1 μg/ml) induced higher IFNγ and IL2 secretion. This effect was independent from the molecular characteristic of the CAR backbone. In parallel we measured the CAR T cell proliferation in a serial killing assay, where we cultured 2×105 HER2 CAR T cells in the presence 1 μg/ml HER2-Fc target antigen for three days. On day 3 the CD3 positive population was counted by flow cytometry and re-challenged on fresh target molecules. We have confirmed our previous result suggesting that CD28z CAR T cells induce better proliferation on HER2 positive targets, but 41BBz CAR T cells are superior in long-term persistence. In summary, we show here that CD28 CARs induce better early activation and proliferation of T cells while expression of 41BB CARs results in longer persistence.