A crude preparation, inhibiting trypsin (T), chymotrypsin (C) and papain (P), was isolated from the culture filtrate of Actinomyces sp. 9 by butanol extraction. Ion-exchange high-performance liquid chromatography (HPLC) of this preparation on a Mono S column resulted in the separation of two inhibitory fractions: one active against T (TI-9) and the other active against C and P (CPI-9). These two fractions, having the same inhibitory specificity, were also obtained from the crude extract by size-exclusion HPLC on a Superose 12 column. This method proved to be better in terms of the purity of the fractionated inhibitors than ion-exchange chromatography. Further purification of TI-9 and CPI-9 was achieved by using reversed-phase HPLC on a PEP RPC column. In this case, TI-9 was obtained as an apparently homogeneous peak. Studies on the physicochemical properties of the purified inhibitors showed them to be small molecules, similar in hydrophobicity, pH and thermal stability, but differing in solubility. Amino acid and spectral analyses provided the peptidic structure of TI-9 and gave a molecular weight of 1643, while parallel analyses of CPI-9 showed that it lacks the common amino acids.