A collagenolytic enzyme, with a molecular weight of 58,000 daltons and isoelectric point of 5.1, was isolated and purified from the venom of the rattlesnake Crotalus atrox by Sephadex G-100 gel filtration followed by chromatography on DEAE-Bio-gel A. The enzyme released alpha-chains from beta-chains of the native collagen and cleaved in the helical region similar to other animal collagenases. The enzyme also hydrolyzed the PZ-peptide, however, it did not hydrolyze synthetic substrates for serine protease (such as TAME or ATEE). The enzyme had no hemorrhagic activity. Immunocross-reactivity suggested that only the venom from Crotalidae contain the enzyme.