There are few reports of HIV-1 protease inhibitors from natural sources. Consequently, we developed a solid-phase immunoassay designed to detect the presence of HIV-1 protease inhibitors in fermentation broths. The assay is based on a recombinant protein consisting of Escherichia coli β-galactosidase fused to a portion of the HIV-1 gag polyprotein containing the p17~p24 cleavage site (acting as a specific substrate for HIV-1 protease) and a very specific monoclonal antibody immobilized on microtiter plates that binds the fusion protein in the gag region only if it has not been cleaved by HIV-1 protease. More than 12,000 strains (70% actinomycetes, 30% fungi) were screened, yielding 31 positive activities after retesting (24 from Streptomyces, 4 from Micromonospora, 2 from Nocardia, 1 fungal). The activity from Streptomyces strain GE16457 was isolated and characterized as belonging to the MAPI complex of α and β epimers, identified by FAB-MS, ¹H and ¹³C NMR. Hydrolysis and HR-GC analysis found L-phenylalanine (consistent with α-MAPI, while β-MAPI contains D-phenylalanine). β-MAPI was also isolated but showed no activity against HIV-1 protease; the oxidation product of active α-MAPI was also inactive, indicating the aldehydic function and configuration of its adjacent atom are crucial for activity. Of 24 positive Streptomyces strains, MAPI complex was found ten times, a common secondary metabolite of Streptomyces.