An antifungal amino acid antibiotic, (S)2-amino-4-oxo-5-hydroxypentanoic acid (RI-331) isolated from Streptomyces sp., inhibited the biosynthesis of protein to a greater extent than that of RNA or DNA in growing Saccharomyces cerevisiae cells. Polypeptide biosynthesis in a cell-free system from the yeast was refractory to the antibiotic, suggesting the possibility that the biosynthesis of one or more amino acids might be inhibited. Intracellular amino acid pools, particularly those of methionine, isoleucine and threonine were significantly reduced when yeast cells were incubated in the presence of RI-331. Consistent with this, the growth-inhibitory activity of RI-331 was markedly reversed by the addition of these amino acids into the growth medium, and an even greater effect was exerted by homoserine which works as a common metabolic precursor for these amino acids in yeasts. It looks likely therefore that the inhibition of biosyntheses of some or all of these amino acids by RI-331 is primarily responsible for overall inhibition of protein biosynthesis in yeasts, ultimately leading to cytostasis. This possible mechanism of RI-331 action appears to explain favorably the selective toxicity of the antibiotic against yeasts, since mammalians lack enzymatic systems for synthesizing methionine, isoleucine and threonine which are required as essential amino acids for growth.