The source of early production of sn-1,2-diacylglycerol (DAG) has for a long time been exclusively linked to hydrolysis of phosphatidylinositol 4,5-diphosphate, which on receptor activation is hydrolyzed into DAG and inositol 1,4,5-trisphosphate. We have investigated the origin of lipid sources of DAG production in smooth muscle cells, in response to contraction induced by peptide agonists. We have performed a quantitative analysis of the molecular species of DAG formed in relation to the known molecular composition of parent phospholipids. The molecular species of phospholipids are sufficiently unique that the phospholipid origin of DAGs and its quantitative contribution to their formation can be measured by HPLC. Cell suspensions (10-15 × 10⁶ cells/ml) from the circular muscle of rabbit rectosigmoid were incubated in the presence of the contractile peptide agonist bombesin (BB) at 10⁶ M. Reactions were stopped at different time intervals from 30 s to 4 min. DAGs were extracted, purified by TLC, and benzoylated with benzoic anhydride. The benzoylated DAGs were first purified by TLC and then by normal phase HPLC before they were injected onto a reverse-phase column and eluted isocratically. Furthermore, phospholipids in the lipid extract [phosphatidylinositol (PI), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylethanolamine (PE)] were purified by TLC and similarly analyzed after hydrolysis to DAGs with phospholipase C (PLC). The DAG molecular species profiles for PI, PC, PS, and PE were all unique. Contraction of cells with BB gave noticeable increases (17-55%) in newly formed DAGs. The major phospholipid source of the newly formed DAGs at 30 s was only ~30% from PI, and the remainder was from PC. In contrast, after 4 min of BB stimulation, a decrease was seen in newly formed DAGs in the peak specific for PI hydrolysis. The data suggest that BB-induced contraction by activation of PLCs results in hydrolysis of different phospholipids. The DAGs formed as a result are qualitatively and quantitatively distinct. This could be the basis for the kinetically different pattern of sustained contraction observed with BB.