Increased phospholipase A₂ (PLA₂) activity was measured in cytosolic fractions of lungs from sheep exposed to smoke from burning cotton or to synthetic smoke consisting of carbon and acrolein, a cotton smoke toxin. Three peaks of PLA₂ activity were identified by heparin-Sepharose chromatography. The heparin-nonbinding PLA₂ activity was twofold higher in the extracts from lungs exposed to smoke than in normal lungs. This activity was identified as the group IV 85-kDa cytosolic PLA₂ (cPLA₂). The activities of the forms of PLA₂ that bound to heparin did not change after smoke exposure. Those activities showed a pH optimum of 9.0, required a millimolar Ca²⁺ concentration for full activity, and were inhibited by 5 mM dithiothreitol. One activity eluted at an NaCl concentration typical for group Ib and V PLA₂ and had the expected substrate specificity. The other form of lung PLA₂ that bound heparin was a group II PLA₂. Lung myeloperoxidase activity increased progressively with increased exposure to smoke. cPLA₂ was identified in sheep neutrophils. With 30 breaths of smoke exposure, there was an increase in cPLA₂ activity without a difference in immunoreactivity on Western blot, indicating that the increased activity was not due to increased amounts of protein. In conclusion, smoke induces increases in resident lung cell cPLA₂ activity that is likely responsible for eicosanoid production, leading to lung inflammation and bronchoconstriction.