Anisomycin is a basic antibiotic which shows good biological activity against Trichomonas vaginalis and Entamoeba histolytica. It is obtained by fermentation of various Streptomyces species, and its structure has been shown to be 2-p-methoxybenzyl-3-trans-acetoxy-4-hydroxypyrrolidine (I). The antibiotic may be extracted with chloroform from filtered broth at pH 9.0. It is recovered as white crystalline solid, and is usually accompanied by some deacetylanisomycin (II). Since the acetoxy function of anisomycin is readily hydrolyzed, it was not immediately clear whether the deacetylanisomycin (II) which accompanied the antibiotic was a product of hydrolysis during the isolation procedure or a prior fermentation product. However, a study of the fermentation showed the deacetylanisomycin (II) accumulated in the broth during the logarithmic phase of growth (Figure 1) and was converted into anisomycin during the phase of assimilation. Most of the anisomycin was produced during the third and fourth days of the fermentation when acetic acid had begun to accumulate in large quantities in the medium. The structure of anisomycin (I) suggests that it might be derived from either tyrosine, phenylalanine, or proline. Labeled amino acids and aliphatic acids were added to the fermentation medium and specific activity of the isolated antibiotic was determined. Usually, the crude antibiotics isolated from the culture medium were hydrolyzed to deacetylanisomycin (II) which was then purified by partition chromatography. Occasionally it was advantageous to isolate anisomycin (I). The results of these fermentations are shown in Table I. We conclude that the pyrrolidine ring of anisomycin is not derived from proline, but that the major precursor is tyrosine. Analysis of the soy meal, which was the only source of amino acids for the fermentation, showed that it contained 3.1% by weight of tyrosine, whereas phenylalanine was present in 4.8%. Thus, although the quantities of these amino acids in the fermentation medium are approximately the same, the tyrosine was incorporated 40 times more efficiently than the phenylalanine. Other amino acids incorporated in significant amounts were glycine and methionine. Labeled acetate was utilized, but the degree of incorporation was erratic, probably owing to dilution by the large amounts of acetic acid produced in the fermentation. Malonate was not utilized to any significant degree.