Basal defined medium (BDM) containing vitamins, minerals and seven amino acids--(/L) tryptophan 0.1 g, methionine 0.2 g, valine 0.3 g, isoleucine 0.3 g, proline 0.3 g, leucine 0.4 g and cysteine 0.5 g--which appeared to be essential for good growth of Clostridium difficile was prepared. Addition of glycine 0.2 g/L and threonine 0.4 g/L to BDM produced better growth of strain VPI 10463, and this defined medium was designated minimum amino acid-defined medium (MADM). Production of toxins A and B by strain VPI 10463 in 6 x MADM containing (/L) tryptophan 0.6 g, methionine 1.2 g, valine 1.8 g, isoleucine 1.8 g, proline 1.8 g, leucine 2.4 g, cysteine 0.5 g, glycine 0.2 g and threonine 0.4 g, was much greater than in MADM. Toxin production by 20 C. difficile strains was examined in two defined media--6 x MADM and complete amino acid-defined medium (CADM) containing 18 amino acids--and one complex medium, modified brain heart infusion medium (m-BHI). Simultaneous production of toxins A and B by all test strains was demonstrated in m-BHI and the two defined media. It was also shown that 6 x MADM was generally better than CADM and as effective as m-BHI for stimulating toxin production by 13 strains. This defined medium would be useful for studies on the physiology, metabolism and pathogenicity of C. difficile.