Previously, specific inhibitors of acid proteases termed pepstatins (A to H) and pepstanones (thought to be derived from pepstatins by oxidative decarboxylation) were obtained from actinomycetes. In this study, three additional pepstatins—Bu, Pr, and Ac (with R groups being butyl, propionyl, and acetyl, respectively)—were isolated from the culture broth of Streptomyces parvisporogenes strain MD494-A1 (isolated from soil in Okusawa, Setagaya-ku, Tokyo, Japan). The strain's cultural characteristics (chromogenic type, smooth spores, growth and aerial mycelium colors, starch/protein hydrolysis, carbon source utilization) were summarized. The pepstatins were produced via rotary shaking culture in a medium containing glucose, starch, Polypeptone, meat extract, and minerals, with maximum production after 60-70 hours of incubation. Isolation and purification involved activated carbon adsorption (eluted with methanol at pH 7.0), carbon column chromatography (70% propanol), Amberlite XAD-2 column chromatography (60% methanol), and silica gel column chromatography (chloroform-methanol-acetic acid for separating Ac from Bu/Pr; n-butanol-pyridine-acetic acid-water for separating Bu and Pr). Structural identification: Hydrolysis revealed fatty acid components (methyl normal butyrate for Bu, methyl propionate for Pr, methyl acetate for Ac) and amino acids (alanine, valine, 4(s)-amino-3(s)-hydroxy-6-methylheptanoic acid in a 1:2:2 molar ratio). Mass spectrometry confirmed molecular weights (Bu: 685, Pr: 671, Ac: 657) and amino acid sequences identical to pepstatin A. Elemental analysis validated their chemical formulas. Inhibitory activity assays showed Bu, Pr, and Ac had similar activity against pepsin and cathepsin D as pepstatins A, B, F, and G; however, their activity against renin increased with the number of carbon atoms in the fatty acid moiety.