Spores of pathogenic Clostridium perfringens and Clostridium difficile must germinate in the food vehicle and/or host's intestinal tract to cause disease. In this work, we examined the germination response of spores of C. perfringens and C. difficile upon incubation with cultured human epithelial cell lines (Caco-2, HeLa and HT-29). C. perfringens spores of various sources were able to germinate to different extents; while spores of a non-food-borne isolate germinated very well, spores of food-borne and animal isolates germinated poorly in human epithelial cells. In contrast, no detectable spore germination (i.e., loss of spore heat resistance) was observed upon incubation of C. difficile spores with epithelial cells; instead, there was a significant (p < 0.01) increase in heat-resistant spore titers. In C. perfringens, the highest spore germination response observed with the HT-29 cell line, might be in part, due to the expression of germination factor with peptidoglycan cortex hydrolysis activity by HT-29 cells. Collectively, these findings might well have implications in understanding the mechanism of clostridial spore germination in vivo.