A novel serine protease inhibitor SIL8, which was isolated from the culture medium of Streptomyces virginiae and shown to be a member of the Streptomyces subtilisin-inhibitor-like (SIL) inhibitor family by sequence analysis of its amino-terminal region [Taguchi, S., Kikuchi, H., Kojima, S., Kumagai, I., Nakase, T., Miura, K. & Momose, H. (1993) Biosci. Biotech. Biochem. 57, 522-524], is the first SIL inhibitor demonstrated to show marked inhibitory activity toward alpha-chymotrypsin, in addition to strong inhibitory activity toward subtilisin BPN', a common property of inhibitors of the Streptomyces subtilisin inhibitor (SSI) family. In this study, the complete amino acid sequence of SIL8 was determined from the sequence analysis of peptides obtained by specific cleavage at the reactive site and by enzymic digestion. SIL8 was shown to exist as a dimer protein, each subunit of which was composed of 111 amino acids, and to have less than 50% similarity with other SSI-family inhibitors, indicating its most distant relationship to other members of this family. Insertion of two residues was observed in the flexible loop region of SIL8, and amino acid replacements were found not only on the molecular surface but also in the beta-sheet and hydrophobic core, suggesting that packing rearrangements of the side chains may occur in these regions to maintain the tertiary and quaternary structures. The inhibitor constants Ki obtained using synthetic substrates are 92 pM for subtilisin BPN' and 11 nM for alpha-chymotrypsin. The P1 site was was identified as methionine, which was in good agreement with the substrate specificity of alpha-chymotrypsin. SSI, which also possesses a methionine residue at the P1 site, inhibits alpha-chymotrypsin poorly (inhibitor constant, 4.0 microM). Such a difference in the inhibitory properties of SIL8 and SSI toward alpha-chymotrypsin is discussed on the basis of the structures of the inhibitors.