Protein transduction domain (PTD) from HIV-1 TAT protein has been reported to translocate across the mammalian cell membrane, also as a part of fusion proteins. However, the true nature of TAT-mediated intercellular spreading is still under debate because it has been claimed to be a fixation artifact. To study the spreading of TAT fusion proteins and their potency to enhance thymidine kinase/ganciclovir (HSV-TK/GCV) cancer gene therapy, we constructed a novel triple fusion protein containing TAT PTD, HSV-TK and green fluorescent protein (TAT-TK-GFP). This fusion protein has three functional domains in the same polypeptide, allowing reliable determination of the relationship between transduction rate and cell killing efficiency. TAT-TK-GFP was cloned into a lentivirus vector and used for analyses of TAT-mediated protein translocation and enhancement of HSV-TK/GCV cytotoxicity. The triple fusion protein was expressed correctly in vitro, but cell-to-cell translocation was not observed in rat glioma cells (BT4C). However, TAT-TK-GFP made BT4C and SKOV3. ip1 (human ovarian carcinoma) cells significantly more sensitive to ganciclovir than TK-GFP, whereas the effect in PC-3 human prostate carcinoma cells was more subtle. It was also observed that growth in lower serum concentration (2.5-5%) abolished the enhancement in BT4C cells, suggesting that high proliferation rate is one of the factors that contribute to TAT PTD-mediated enhancement of cytotoxicity. In summary, our results indicate that TAT PTD fusion proteins do not translocate intercellularly at detectable levels, but enhancement of the HSV-TK/GCV cytotoxicity can be detected in rat and human tumor cell lines in vitro.