A broad host range multiresistance plasmid pPK237, originating from Pseudomonas aeruginosa mediates high-level resistance to gentamicin and tobramycin. It was found to code for two gentamicin modifying enzymes, which from their substrate profile by radioenzymatic assay were characterized as aminoglycoside acetyltransferase AAC(3)-I and aminoglycoside adenylyltransferase AAD(2"). The two enzymes were studied after purification from an Escherichia coli K12 host. The two gentamicin-modifying enzymes coded by pPK237 were completely separated by DEAE chromatography. The purification (126 fold) of the acetyltransferase was achieved by (NH₄)₂SO₄ precipitation, DEAE chromatography and affinity chromatography. The purification of the adenylyltransferase was performed by affinity chromatography directly after (NH₄)₂SO₄ precipitation. Both purified enzyme preparations showed a single protein band on disc electrophoresis. The Km for gentamicin C₁ of the acetyltransferase was 0.066 mM. The amino acid analysis of the acetyltransferase coded by pPK237 showed a different aminoacid composition than that of the gentamicin acetyltransferase AAC(3)-I purified by WILLIAMS and NORTHROP17). The acetyltransferase after DEAE chromatography is stable for many months at -20°C, while the adenylyltransferase after purification is highly unstable; it shows enzymatic activity only in the presence of Mg⁺⁺.