Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.