Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 .mu.mol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56.degree.C, 10 h at 37.degree.C, and 5 days at 4.degree.C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI''s of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.