Thirty-eight chromogenic substrates were used to study the specificity of the proteolytic enzymes of seven species of dermatophytes and three related keratinolytic soil fungi. The source of enzymes were cultivation fluids from cultures of the fungi grown on human hair. The overall specificity of the enzymes of all the keratinolytic fungi was very similar. Aminoacyl- and dipeptidyl-4-nitroanilides, substrates of aminopeptidases and dipeptidyl aminopeptidases respectively, were poor substrates compared to aminoterminally blocked oligopeptidyl derivatives. Of the latter, the best substrates were those with phenylalanine, leucine, alanine, methionine or arginine (i.e. amino acids with hydrophobic or basic side chains) in the P1 position. Of the amino acids in the P2 position, proline was the most effective at accelerating the hydrolysis of the respective substrates. Positions P3 and P4 and even the aminoterminal protecting group were also of importance. The specificity profiles of the proteolytic enzymes corresponded best to those of some well characterized serine proteinases (chymotrypsin, elastase).