The present study intended to verify activities of cysteine proteinase of Pneumocystis carinii from rats and to purify the enzyme. In order to exclude the contamination of host-derived enzymes, concentrates of P. carinii was primarily treated with a mixture of proteinase inhibitors before lysis of P. carinii. A 68-kDa cysteine proteinase was finally purified from the crude extract of P. carinii by 4 sequential chromatographic methods. The enzyme showed an optimal activity at pH 5.5 in 0.1 M sodium acetate, and its activity was specifically inhibited by L-trans-epoxysuccinylleucylamido (4-guanidino) butane (E-64) and iodoacetic acid, suggesting that the enzyme is a cysteine proteinase. The 68-kDa proteinase weakly digested macromolecules such as collagen, hemoglobin and fibronectin. The present study demonstrated the activity of cysteine proteinase at the 68-kDa band of P. carinii, and purified and characterized the molecule.