In 1970, Umezawa et al. isolated pepstatin, a specific pepsin inhibitor, from cultured broths of Streptomyces testaceus Hamada et Okami and Streptomyces argenteolus var. toyonakensis. Pepstatin is the N-iso-valeryl derivative of a pentapeptide composed of one mole of L-alanine, two moles of L-valine and two moles of 4-amino-3-hydroxy-6 methylheptanoic acid, with its amino acid sequence determined by mass spectrometry. In this communication, we report new pepsin inhibitors—pepstatins B (II) and C (III), and pepstanone A (IV)—produced by the same pepstatin-producing Streptomyces, assigning the former pepstatin the name pepstatin A. Pepstatins B and C are N-n-caproyl and N-iso-caproyl derivatives of the pentapeptide, with structures confirmed by mass spectrometry and hydrolysis studies, and their pepsin-inhibitory activity is comparable to that of pepstatin A. Pepstanone A, a minor component with a keto group, has a molecular formula C33H61O7N5, a structure elucidated via elemental analysis, mass spectrometry, and chemical degradation, and exhibits 78–86% of the activity of pepstatin A. Analysis of peptone and casein media reveals remarkable medium-dependent differences in the yields of these compounds.