Supplementation of a carbohydrate-free minimal medium with a high level (100 mM) of histidine, methionine, valine, isoleucine, proline and leucine, in particular isoleucine, markedly increased toxin production by Clostridium difficile VPI 10463. The effect of isoleucine was further examined. Increasing the concentration of isoleucine from 20 to 100 mM remarkably increased toxin production, while bacterial growth decreased gradually. Amino acid analysis of the culture revealed that, at 100 mM isoleucine, consumption of isoleucine was remarkably increased. During the incubation period when toxin titers increased markedly but bacterial growth was declining, isoleucine, leucine and cysteine were taken up preferentially and alanine and cystathionine, which were not found at 1 mM isoleucine, were produced in large quantities. These findings suggest that isoleucine may play an important role in toxin production by C. difficile and that alanine and cystathionine production may be co-regulated with the toxin production in the absence of fermentable carbohydrates.