Diacylglycerol kinase is though to play a central role in the metabolism of diacylglycerol second messengers in agonist-stimulated cells. A series of diacylglycerol analogs were tested for their ability to act as substrates or inhibitors of diacylglycerol kinase with the goal of determining the substrate specificity of the enzyme, and of discovering inhibitors. Screening of these compounds was performed using a partially purified diacylglycerol kinase from pig brain. Modified assays for this enzyme using co-sonicated mixtures of diacylglycerol and anionic phospholipids were developed. This enzyme was found to be quite specific for sn-1,2-diacylglycerol (KM 24 microM for dioctanoyl-glycerol). Among the analogs investigated, only 1,2-dioctanoyl-2-amino-1,3-propanediol was utilized at a significant rate. Two analogs, dioctanoylethylene glycol (KI 58 microM) and 1-monooleoylglycerol (KI 91 microM), were potent inhibitors in vitro. These compounds were tested for effects on diacylglycerol formation and metabolism in thrombin-stimulated human platelets. Dioctanoylethylene glycol inhibited diacylglycerol phosphorylation in platelets (70-100% at 100 microM) leading to a longer-lived diacylglycerol signal. This compound may be a useful tool for studies of diacylglycerol kinase in other cell types. 1-Monooleoylglycerol treatment elevated diacylglycerol levels up to 4-fold in unstimulated platelets and up to 10-fold in thrombin-stimulated platelets. The implications with regard to the pathways of diacylglycerol metabolism in human platelets are discussed.