Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.