Phosphatidylcholine (PC) synthesis by alveolar type II cells, as an indicator for the production of pulmonary surfactant, was studied after a 4-h exposure of rats to 4 mg ozone/m3 (2 ppm). Lung ravage fluid analysis after exposure revealed significant increases in proteins, which is indicative for pulmonary injury. When type II cells were isolated immediately and thereafter cultured for 20 h, the rate of PC synthesis in cells derived from ozone-exposed rats was not significantly different from that in cells from unexposed controls. Yet, a decreased rate of PC synthesis was observed when these cells were subsequently exposed to ozone in vitro. The activity of the enzyme glycerolphosphate acyltransferase (GPAT) was slightly enhanced in cultured type II cells isolated from ozone-exposed rats, while the lysophosphatidylcholine acyltransferase (LPCAT) activity was unchanged. However, ozone exposure of rats did result in a significant decrease of PC synthesis when measured in freshly prepared type II cell suspensions, although both GPAT and LPCAT activities were not affected. It is concluded that a decrease in pulmonary surfactant related PC synthesis after ozone exposure of rats can be demonstrated in freshly isolated type II pneumocytes. Cultured type II cells from exposed rats lack this effect and are therefore less useful to study changes in phospholipid biosynthesis after in vivo ozone exposure. The data on in vitro ozone exposure of cultured type II cells, however, support the view that ozone may impair pulmonary surfactant production.