Increased cAMP by stimulation of adenylyl cyclase with forskolin or by beta-adrenoceptor activation with isoproterenol increased phospholipase D (PLD) activity in tracheal smooth muscle strips. PLD activity was measured by the accumulation of phosphatidylethanol. A linear increase in the concentration of phosphatidylethanol was observed over 20 min in muscle strips treated with either forskolin or isoproterenol. Cholinergic stimulation with acetylcholine (ACh), by contrast, caused a rapid increase in phosphatidylethanol followed by a slow decline in the concentration of phosphatidylethanol from 5 to 20 min in the continued presence of ACh. Concomitant treatment with ACh and either forskolin or isoproterenol eliminated the rapid increases in phosphatidylethanol associated with ACh treatment. The response to forskolin or isoproterenol was not influenced by ACh. Inhibition of protein kinase C with calphostin C or bisindolylmaleimide I had no effect on isoproterenol- or forskolin-stimulated PLD activity but inhibited ACh-activated PLD activity. Protein kinase A (PKA) inhibitors H-89 and KT5720 significantly decreased forskolin- and isoproterenol-mediated activation of PLD activity. PKA inhibition also eliminated inhibition of ACh-stimulated PLD activity by forskolin or isoproterenol. Activation of adenylyl cyclase by forskolin or by isoproterenol caused increased phosphorylation of phospholipase C-beta(2) isoform and reduced the formation of inositol phosphates after ACh stimulation of muscarinic receptors. These results suggest that increasing the concentration of cAMP activates PLD via activation of PKA and that the increased activity of PKA also inhibits cholinergic stimulation of PLD, in part at least by inhibiting the activation of phospholipase C by ACh.