The antioxidant, butylated hydroxytoluene (BHT), causes lung toxicity in mice followed by regenerative repair, and can also modulate the development of carcinogen-induced lung adenomas. We are investigating changes in pulmonary biochemistry following BHT treatment in order to understand the mechanisms of BHT-induced pulmonary regenerative repair. BHT administration lowered cytosolic Ca2+-activated neutral protease (calpain) activity, increased the activity of the endogenous calpain inhibitor, calpastatin, increased the extent of photoincorporation of 8-N3-[32P]cAMP into a Mr 37,000 proteolytic product derived from cAMP-dependent protein kinase regulatory (R) subunits, and increased membrane-associated protease activity. All of these changes were dependent on the BHT dosage; the altered proteolytic activities occurred at a dose lower than that which caused observable lung toxicity as assessed by the lung weight/body weight ratio. Decreased cytosolic calpain activity was detectable within 1 day after BHT administration, was lowest at 4-7 days, and had not returned to control levels by Day 21, a time when normal lung morphology had been regained. The decrease in calpain activity cannot fully be accounted for by increased calpastatin activity; upon separation of these proteins by DEAE chromatography, the amount of calpain activity from BHT-treated mice remained lower than the corresponding peak from control mice. Increased photolabeling of the Mr 37,000 protein began at 1 day and continued to increase up to 4 days after BHT. All of the cytosolic changes preceded the increased particulate proteolytic activity by 1-2 days. R-subunits which have dissociated from their catalytic subunits are more susceptible to degradation by calpain, but BHT treatment did not enhance subunit dissociation as determined by the elution profile of 8-N3-[32P]cAMP-labeled R-subunits following DEAE chromatography. A large percentage of the particulate protease activity was inhibited by calpastatin, leupeptin, and E-64, all of which are known to inhibit calpain activity; this suggested that calpain accounted for most of this activity. Changes in the activities of proteases which catalyze limited proteolysis reactions may play an important role in the repair of acute lung injury.