A novel actinomycin (Act SG 3) from a strain of Streptomyces galbus var. C-72, as well as actinomycin D (Act D) were found to act as competitive inhibitors of serine proteinases from microorganisms. The inhibitory properties of Act SG 3 and Act D are compared with these of other peptide antibiotics, namely bacitracin A (Bac A) and gramicidin S (Gr S). The last compound has only a weak inhibitory effect. The following order of affinity for the four peptide antibiotics towards subtilisin DY and proteinase K was observed: Bac A > Act D > Act SG 3 = Gr S. The affinity towards thermitase changes as follows: Act SG 3 = Act D > Bac A>Gr S.