Serum coagulation factor X (FX) is proposed to play a major role in adenovirus tropism, promoting transduction by bridging the virus to cell-surface heparan sulfate proteoglycans (HSPGs). Both murine FX and human FX increased transduction by Ad.CMVfLuc, an adenovirus vector, in murine hepatocyte-like cells and human hepatocarcinoma cells. In contrast, only hFX increased transduction of several non-hepatic cancer cell lines and Chinese hamster ovary (CHO) cells. Not only was mFX unable to promote transduction in these cells, it competitively blocked hFX-enhanced transduction. Competition and HSPG digestion experiments suggested mFX- and hFX-enhanced transduction in hepatocyte-derived cells, and hFX-enhanced transduction in epithelial cancer cells were dependent on HSPGs. Ad·hFX-mediated transduction of CHO mutants unable to produce HSPGs was also curtailed. Hepatocyte-derived cells expressed substantially more HSPGs than the cancer cell lines. Dose-response curves and heparin-Sepharose binding suggested Ad·hFX has greater affinity for HSPGs than does Ad·mFX. In coagulation factor-depleted mice hFX also had enhanced ability, compared with mFX, to reconstitute hepatic adenovirus transduction. The results suggest that differences in Ad·hFX and Ad·mFX affinity to HSPGs may result in differences in their ability to enhance adenovirus transduction of many cells. These findings may have implications for murine models of adenovirus vector targeting.