Certain chloromethyl ketone (CH 2Cl) protease inhibitors diminish PMA-stimulated (phorbol myristate acetate) superoxide (O 2-) release by both human alveolar macrophages (HAM) and polymorphonuclear leukocytes (PMN). Additionally, these compounds diminish glutathione reductase activity (GSSR) and intracellular levels of reduced glutathione (GSH). Inhibitory profiles of these molecules were similar for each of the measured cell products, i.e., TPCK = Z Gly-Leu-Phe-CH 2Cl > TLCK > Meo Succ-(Ala) 2-Pro-Val-CH 2Cl. Because of Meo Succ-(Ala) 2-Pro-Val inhibited GSSR in cell sonicates but not in intact cells, it appears that all these effects are closely related to the cell penetration by these CH 2Cl compounds. We conclude that (1) inhibition of O 2- release in PMN and HAM by CH 2Cl does not necessarily implicate a surface protease in O 2- production, (2) some of these compounds certainly impair the intracellular glutathione redox system necessary for the respiratory burst, and (3) only the nonpenetrating inhibitor Meo Succ-(Ala) 2-Pro-Val-CH 2Cl can be used to distinguish injury caused by extracellular protease (elastase) from that caused by superoxide anion.