Staurosporine (STP), a potent protein kinase C inhibitor first isolated in 1977, had its biosynthesis limited to early steps (e.g., tryptophan incorporation into aglycone) until HOEHN et al. (1995) hypothesized O-methylation of 3'-demethoxy-3'-hydroxystaurosporine (DMSTP) as the final step. This study describes the detection of DMSTP-O-methyltransferase catalyzing this step. The enzyme was demonstrated by biotransformation of DMSTP to STP using STP-aglycon producing mutant M13 and activity assays in crude cell extracts of Streptomyces longisporoflavus strains (PHT3, SG71, R19/col15, M13), while DMSTP-producing mutant M14 lacked the activity. S-adenosyl-methionine (SAM) served as the methyl donor, with S-adenosyl-L-homocysteine (SAH) strongly inhibiting the enzyme. Low-concentration tryptophan (>2-fold activation at 0.2mM) while high concentrations and other aglycone precursors showed moderate inhibition. These results confirm that DMSTP methylation is the last step in STP biosynthesis.