Puromycin 2"-N-acetyltransferase was isolated from cell extracts of puromycin-producing Streptomyces alboniger KCC S-0309 by ammonium sulfate fractionation, heat treatment to eliminate contaminant proteins and chromatography on DEAE-Toyopearl 650S. After PAGE (polyacrylamide gel electrophoresis) of the final fraction, a single protein band corresponding to puromycin 2"-N-acetyltransferase was detected. The molecular weight of the enzyme determined by SDS-PAGE and Sephadex G-150 chromatography was about 21,000 and 85,000, respectively, suggesting that the enzyme consisted of four subunits. The isoelectric point and the optimum pH for reaction were 6.2 and 7.7, respectively. The Km values for puromycin and acetyl coenzyme A were 40 microM and 67 microM, respectively. The enzyme was thermostable up to 70 degrees C for 12 minutes. It was shown, by using an in vitro protein synthesizing system from a puromycin-susceptible organism S. flavotricini subsp. pseudochromogenes V-13-1, that the isolated puromycin 2"-N-acetyltransferase could protect polyphenylalanine synthesis from inhibition by puromycin.