Recombinant adeno-associated viruses (rAAVs) are becoming more commonly used in clinical trials involving gene therapy. Additionally AAV-based drugs have already been registered. Gene therapy aims to increase transduction efficiency, increase selectivity and reduce side effects. One approach to achieve this is the use of physical factors, such as temperature or more specifically, hyperthermia, which is already utilized in oncology. The aim of the present study was to investigate the effect of hyperthermic conditions (40°C and 43°C) on the rAAV transduction efficiency of ovarian cancer cells (Caov-3 and NIH:OVCAR-3) and non-cancerous cells (AAV-293). The present study was designed to identify functional associations between the level of gene transfer and the expression of representative genes for rAAV transmission (AAVR (AAV receptor), heparan sulfate proteoglycan (HSPG) 1 and HSPG2) and heat shock proteins (HSPs). The expressions of selected genes were measured via reverse transcription-quantitative PCR and cell adhesion/invasion chamber tests were also performed. The results revealed that ovarian cancer cell lines were more efficiently transduced with rAAV vectors at an elevated temperature. Additionally, the expression patterns of AAVR, HSPG1 and HSPG2 genes were different between the tested lines. The expression of certain receptors in ascites-derived NIH:OVCAR-3 ovarian cancer cells was higher compared with tumor-derived Caov-3 cells at 37, 40 and 43°C, which indicates a higher transduction efficiency in the formerly mentioned cells. Ascites-derived ovarian cancer cells were characterized by high expressions of HSP40, HSP90 and HSP70 families. Lower levels of HSP expression were demonstrated in less-effectively transduced Caov-3 cells. Furthermore, expressions of the examined genes changed with increasing temperature. The results indicated that temperature-dependent transduction is associated with the expression of the rAAV receptor and HSP genes. The results of the current study may aid the design of effective protocols for ovarian cancer gene therapy.