Changes in diacylglycerol kinase (DG kinase) activity in carbachol (CCh)-stimulated guinea pig taenia coli were investigated. In phosphorus-32 ([32P]Pi)- and exogenous substrate of DG (dioctanoylglycerol; diC8)-prelabeled guinea pig taenia coli, diC8 was phosphorylated by DG kinase to [32P]dioctanoyl-phosphatidic acid ([32P]diC8-PA). CCh increased the accumulation of both [32P]diC8-PA and endogenous [32P]PA in a time- and dose-dependent manner. CCh-induced increase in [32P]diC8-PA was inhibited by atropine. These findings indicated the activation of DG kinase by muscarinic receptor stimulation. CCh-induced [32P]diC8-PA accumulation was dependent on intracellular calcium concentration. However, a KCl-induced increase in intracellular calcium, without receptor stimulation, was ineffective. Moreover, treatment with phorbolester also increased the accumulation of [32P]diC8-PA in KCl-treated tissues. These findings suggest that activation of DG kinase may require both an increase in intracellular calcium and protein kinase C (PKC) activation. In an mixed micellar assay system, DG kinase activities were distributed in both membrane and cytosolic fractions. Treatment of the tissue with CCh increased the activity in membrane fraction and decreased the cytosolic fraction without affecting total DG kinase activity. These findings suggested that DG kinase might be translocated from the cytosol to the membrane by CCh-stimulation. Treatment with PKC inhibitor blocked CCh-induced DG kinase translocation, and phorbolester induced the translocation only in intracellular calcium accumulated tissue. Considering these results, CCh-induced DG kinase activation appears to involve DG kinase translocation from the cytosol to the membrane in association with both PKC and intracellular calcium concentration.