Our earlier studies have indicated the presence of diacylglycerol kinase activity in rat brain cytosol as well as subcellular membrane fractions (Strosznajder et al.: Neurochemistry International 8(2):213-221, 1986). There is much evidence indicating the release of diacylglycerols due to stimulation of polyphosphoinositide hydrolysis by hormones and receptor agonists. In turn, diacylglycerols have been linked to a second messenger role for activation of protein kinase C. The present study tests the ability of free fatty acids and acyl-coenzyme A (acyl-CoA) to regulate diacylglycerol kinase activity. In a system containing brain cytosol and microsomes, addition of oleic acid (0.5 mM) resulted in large stimulation of diacylglycerol kinase activity as well as some translocation of the enzyme from cytosol to microsomes. On the other hand, oleoyl-CoA (0.1 mM), but neither palmitoyl-CoA nor arachidonoyl-CoA, was effective in translocation of the diacylglycerol kinase. Unlike oleic acid, which preferred to associate with membranes, most of the oleoyl-CoA remained in the cytosolic fraction. Since free fatty acids in brain are stringently controlled and are released during ischemic insult, a condition which also elicits the breakdown of polyphosphoinositide to diacylglycerols, results here suggest a plausible mechanism for regulation of diacylglycerol metabolism by free fatty acids and acyl-CoA.