<jats:title>Abstract</jats:title><jats:p><jats:italic>Blasticidin S is a potent antifungal and cytotoxic peptidyl nucleoside antibiotic from</jats:italic> Streptomyces griseochromogenes. <jats:italic>The mixed biosynthesis of the compound is evident from the three distinct structural components: a cytosine base, an amino deoxyglucuronic acid, and</jats:italic> N<jats:italic>‐methyl</jats:italic> β<jats:italic>‐arginine. The blasticidin S biosynthesis gene cluster was cloned from</jats:italic> S. griseochromogenes <jats:italic>and the pathway heterologously expressed in</jats:italic> S. lividans <jats:italic>from a cosmid harboring a 36.7‐kb fragment of</jats:italic> S. griseochromogenes <jats:italic>DNA. The complete DNA sequence of this insert has now been determined and evidence suggests a contiguous 20‐kb section defines the blasticidin S biosynthesis cluster. The predicted functions of several open reading frames are consistent with the expected biochemistry and include an arginine 2,3‐aminomutase, a cytosylglucuronic acid synthase, and a guanidino</jats:italic> N<jats:italic>‐methyltransferase. Insight into other steps in the assembly of blasticidin S was evident from sequence homology with proteins of known function and heterologous expression of fragments of the cluster. Additionally, the gene that directs the production of free cytosine</jats:italic>, blsM, <jats:italic>was subcloned and expressed in</jats:italic> Escherichia coli. <jats:italic>Characterization of BlsM revealed that cytidine monophosphate serves as the precursor to cytosine.</jats:italic>