Chemotherapy is regularly used for cancer treatment but often results in the development of drug resistance. The development of drug resistance could lead to differential expression of Adenovirus type 5 (Ad5) receptors as well as to changes in the expression of the transgene from Ad5 vector. We investigated whether there are differences in Ad5–mediated transgene expression between human laryngeal carcinoma (HEp2) cells and corresponding, HEp2-derived, vincristine resistant cell line VK2. We measured the transgene expression from wild type Ad5wtRSVβgal, short fibered Ad5Δ639RSVβgal which infects cells independent of primary Ad5 receptor CAR (coxsackie and adenovirus receptor) and Ad5RGD4CRSVβgal retargeted to αv-integrins. We observed 4,5-fold increase in Ad5wtRSVβgal and Ad5RGD4CRSVβgal transgene expression in VK2 cells comparing to HEp2 cells, while for Ad5Δ639RSVβgal this difference was 10-fold. We found differences between HEp2 and VK2 cells in cell surface expression of Ad5 receptors but these changes were not responsible for observed increased Ad5-mediated transgene expression in VK2 in comparison to HEp2 cells. The Ad5wtRSVβgal attachment and internalisation was 25-30% and 15-30% lower in VK2 than in HEp2 cells, respectively. Finally, Ad5wtCMVβgal differing from Ad5wtRSVβgal ony in promoter that drives transgene expression, showed equal transgene expression in HEp2 and VK2 cells. We conclude that the increased Ad5wtRSVβgal-mediated transgene expression in the VK2 cells is due to the increased RSV promoter activity. Our results point out that (i) drug-resistance may be accompanied with an alteration in promoter activity and (ii) the proper choice of promoter could contribute to a decrease in the vector dose required to achieve a therapeutic effect during tumor gene therapy.