Activation of adenosine A1-, bradykinin- or P2U-receptors on DDT1 MF-2 smooth muscle cells all increased the formation of inositol 1,4,5-trisphosphate and the mobilization of intracellular calcium. All three types of agents could increase [Ca2+]i in the same cell. Activation of the P2U receptor with ATP or UTP produced larger responses than activation of bradykinin- and adenosine A1-receptors, with bradykinin and N6-cyclopentyladenosine. When agonist-stimulated levels of diacylglycerol were determined, all agonists caused biphasic changes of similar magnitudes. If anything, ATP and UTP tended to give larger increases in the second phase of stimulation. Phospholipase D, measured as the formation of phosphatidylethanol in cells labeled with [3H]palmitic acid and activated in the presence of ethanol, was activated similarly as phospholipase C, i.e. ATP or UTP caused the largest increase in phosphatidylethanol formation, followed by N6-cyclopentyladenosine and bradykinin which caused weaker responses. Activation of PLD by P2U receptors was pertussis toxin insensitive. The activation of PLD by the agonists was only weakly affected by a PKC inhibitor, Ro 31-7549 (3-[1-(3-aminopropanyl)-3- indolyl]-4-(1-methyl-3-indolyl)-1H-pyrrole-2,5-dione). In contrast, ATP or UTP did not activate protein kinase C, determined in a permeabilized cell assay using two specific protein kinase C substrates, whereas N6-cyclopentyladenosine and bradykinin caused a substantial activation.(ABSTRACT TRUNCATED AT 250 WORDS)