The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate. For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3. All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3'. This agrees well with the occupancies of these sites by these residues in type I collagen. However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen. Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen. In general, the class II enzymes have a broader specificity than the class I enzymes. However, they are much less active toward sequences containing Hyp in subsites P1 and P3'. Thus, the two classes of collagenases have similar but complementary sequence specificities. This accounts for the ability of the two classes of enzymes to synergistically digest collagen.