A role for phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as a mechanism of alpha 2-adrenergic signal transduction in rabbit tracheal epithelial cells (tracheocytes) was investigated in isolated cells grown in in vitro culture and prelabeled with myo-[3H]inositol (3 microCi/ml) for 72 h. Breakdown of polyphosphoinositides was measured by using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate [PtdIns(4)P], and PtdIns(4,5)P2. Inositol phosphates were separated by ion-exchange column chromatography. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agonists clonidine and 1-(2,6-dichlorobenzylideneamino)guanidine (guanabenz) produced a rapid transient accumulation of inositol trisphosphate and inositol 4,5-bisphosphate and breakdown of [PtdIns(4)P] and PtdIns(4,5)P2. The alpha 2-adrenergic effects were not blocked by the beta-adrenergic antagonist DL-propranolol or by the alpha 1-adrenergic antagonists prazosin and methylurapidil but were inhibited by pertussis toxin and blocked by yohimbine, an alpha 2-adrenergic antagonist. The 50% effective concentration for guanabenz-stimulated inositol trisphosphate generation was right shifted from 0.3 to 0.9 microM by yohimbine. The results provide the first demonstration of alpha 2A-adrenergic activation of pertussis toxin-sensitive PtdIns(4,5)P2-dependent phospholipase C in mammalian tracheocytes. The findings are consistent with previous observations on alpha 2A-adrenergic-mediated activation of NaCl cotransport in these cells.