The regulatory mechanisms of diacylglycerol (DG) kinase activity were studied in guinea pig taenia coli. In an octylglycoside mixed micellar assay system, DG kinase activities were distributed in both membrane and cytosolic fractions. Treatment of the tissue with carbachol (CCh) increased the activity in the membrane fraction and decreased the cytosolic fraction without affecting total DG kinase activity. The Km value of DG kinase in the membrane fraction was unchanged by treatment with CCh, although Vmax was increased. These findings suggest that DG kinase may be translocated from the cytosol to the membrane by CCh-stimulation. Increase in DG content by treatment of tissue with a cell-permeable species of DG, dioctanoyl-sn-glycerol, did not induce DG kinase translocation. Each treatment with protein kinase C (PKC) inhibitor and PKC-desensitization blocked CCh-induced DG kinase translocation; and phorbol ester induced the translocation only in intracellular calcium-accumulated tissues. 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 rather than cellular DG content.