The effect of biotin on toxin production by Clostridium difficile was examined in a defined medium. When toxin production by strain KZ 1647, which was isolated from a healthy adult, was examined in relation to its biotin requirement, it was found that with decreasing concentrations of biotin, bacterial growth was decreased, but production of both toxins A and B were remarkably increased, particularly with 0.05 nM biotin. The time course of production of both toxins in biotin-limited conditions was similar to that in biotin-enriched conditions. The biotin effect on toxin production was also observed in 15 other strains, suggesting that the effect occurs frequently amongst toxigenic C. difficile strains. The biotin effect is discussed in relation to the pathogenesis of C. difficile colitis.