Diacyl glycerol lipase activity has been examined of intracellular and surface membranes isolated from human blood platelets by free flow electrophoresis. Enzyme activity is present on both membranes but is activated at different substrate concentrations (Km 14 microM and 140 microM for intracellular and surface membrane, respectively). Both enzyme activities are stimulated by EGTA and GSH, and inhibited by added Ca2+. The specificity of the intracellular membrane enzyme has been investigated using a range of diacylglycerol substrates differing only in their '2' position fatty acid. Arachidonic acid is clearly the preferred '2' position moiety with activities towards eicosatrienoic, linoleic, oleic and palmitic acid-containing substrates, all substantially lower.