An amino acid antibiotic RI-331, (S)-2-amino-5-hydroxy-4-oxopentanoic acid, produced by a Streptomyces sp., is a novel antifungal agent. Biochemical studies on the mechanism of action of the antibiotic were carried out with a susceptible yeast Saccharomyces cerevisiae. In growing yeast cells, RI-331 inhibited protein synthesis to a greater extent than RNA or DNA synthesis. However, polypeptide synthesis by a cell-free preparation from yeasts was refractory to the antibiotic. The inhibition of protein synthesis by RI-331 was revealed to be due to the depletion of several amino acids such as threonine, methionine, isoleucine and serine in the cellular pool. These results suggest the possibility that some steps in amino acid metabolic pathways, especially those involved in biosynthesis of threonine, methionine and isoleucine, may constitute a primary target of RI-331 action.