In 1970, Umezawa et al. isolated pepstatin (later named pepstatin A), a specific pepsin inhibitor, from Streptomyces testaceus Hamada et Okami and Streptomyces argenteolus var. toyonakensis. This communication reports new pepsin inhibitors—pepstatins B (II) and C (III), and pepstanone A (IV)—produced by the same pepstatin-producing Streptomyces. Pepstatins B and C are the N-n-caproyl and N-iso-caproyl derivatives, respectively, of the pentapeptide (composed of L-alanine, L-valine, and 4-amino-3-hydroxy-6-methylheptanoic acid) in pepstatin A; their structures were confirmed by mass spectrometry, and they showed pepsin-inhibitory activity similar to that of pepstatin A. Pepstanone A, a minor active component, has the molecular formula C₃₃H₆₁O₇N₅ with a keto-group (evidenced by UV, IR, and Brady reaction), its structure (IV) was deduced via hydrolysis, spectral analysis, and mass spectrometry, and it exhibited 78~86% of the pepsin-inhibitory activity of pepstatin A. Additionally, the production of each pepstatin differed remarkably depending on the culture medium (peptone medium vs. casein medium).