Insulin treatment increases the SN-1,2-diacylglycerol (DAG) concentration in skeletal muscle. Because DAG may participate in transmission or modulation of the insulin receptor signal, we examined the effect of insulin on total DAG and on different DAG species in isolated rat hemidiaphragms incubated with 5 mmol/L glucose. Five DAG species (16:0-18:1 omega 9, 16:0-18:1 omega 7, 18:0-18:1 omega 9, 18:0-18:2 omega 6, and 18:1-18:2) were identified and quantified. After a 5-minute incubation with 60 nmol/L insulin, neither total DAG nor a DAG species increased; exposure to insulin for 10 or 20 minutes increased the concentration of total DAG and of several DAG species. Insulin did not increase DAG in muscles incubated without glucose. Two sources for the insulin-mediated DAG increase were considered: phosphatidylcholine (PC) hydrolysis and de novo DAG synthesis from glucose. Concentrations of choline and phosphocholine in muscle were not increased after 10-minute incubations with insulin. However, insulin increased 14C incorporation from [U-14C]glucose into DAG, triacylglycerol (TAG), and total lipids approximately threefold. Okadaic acid (OKA), an inhibitor of phosphoprotein phosphatases 1 and 2A, increased muscle DAG content and synthesis from glucose, similar to the effect of insulin. Doses of OKA or insulin that increased DAG mass greatly exceeded those required for stimulation of glucose transport. The insulin-mediated, relatively slow increase in muscle DAG observed here likely reflects primarily de novo synthesis from glucose. This effect would be downstream of insulin stimulation of glucose transport. However, a possible insulin-mediated, rapid transient increase in muscle DAG content and PC hydrolysis cannot be ruled out by our studies.