Investigations of the biosynthesis of the antitumor antibiotic sparsomycin (1) by Streptomyces sparsogenes have been carried out. Incorporation studies employing (\sp13C)-labeled precursors have shown that the monoxodithioacetal moiety (6) of the antibiotic arises from the step-wise introduction of a thiomethyl group into the S-methyl group of S-methyl-D-cysteine. The methyl group of (6) has its origin in the methyl group of L-methionine, but an experiment utilizing (methyl-\sp3H,\sp35S) -L-methionine has demonstrated that intact incorporation of the thiomethyl group of this precursor does not occur. The results of feeding experiments with (\sp13C)- and (\sp2H)-labeled forms of tryptophan have indicated that the uracil moiety (7) of sparsomycin is derived from the indole nucleus of tryptophan via aromatic ring cleavage followed by recyclization. Preliminary evidence for the intermediacy of N-formyl-anthranilic acid in the conversion of tryptophan to sparsomycin has been obtained.