The biosynthesis of the antitumor antibiotic, sparsomycin, produced by Streptomyces sparsogenes, has been investigated. Incorporation studies employing (\sp13C)-labeled precursors have shown that both L- and D-isomers of S-(methylthio)methyl-cysteine are specifically incorporated into the antibiotic. Furthermore, both L- and D-isomers of S-(methylthio)methylcysteinol are proved to be the advanced intermediates lying beyond S-(methylthio)methylcysteine. S-Methylcysteine is found not to be incorporated intact into the antibiotic; however, an isotopic trapping experiment and preliminary cell-free studies indicate that S-methylcysteine is still quite likely to be an intermediate on the pathway. A very high isotope effect was observed during studies of the loss of hydrogen atoms from the methyl group of S-methylcysteine. The uracil moiety (6) of sparsomycin is found to be derived from the indole fragment of tryptophan. N-Formylanthranilic acid was originally proposed as an intermediate lying beyond tryptophan on the pathway to 6. However, precursor incorporation studies have shown that N-formylanthranilic acid is incorporated by deformylation to anthranilic acid, which is then converted back to tryptophan before incorporation into sparsomycin. Therefore, the originally proposed pathway has to be revised. The final steps in the biosynthesis of uracil moiety are shown to be similar to the biosynthesis of xanthosine monophosphate (XMP).