We have investigated the involvement of specific phospholipase systems and their possible mutual relationship with the mechanism by which atrial natriuretic factor (ANF) increases phosphatidate (PA) and diacylglycerol (DAG) in rat aortic smooth muscle cells (RASMC), one of the major targets of this hormone. Our results indicate that ANF initially stimulates a phosphatidylinositol-dependent phospholipase C (PI-PLC) with a significant increase of DAG, enriched in arachidonate, and inositol trisphosphate (IP3) and then a phosphatidylcholine-dependent phospholipase C (PC-PLC) with formation of DAG, enriched in myristate, and phosphocholine (Pcho). Moreover, ANF stimulates PA formation at an intermediate stage between early and late DAG formation. The transphosphatidylation reaction, as well as its labeling ratio, demonstrate that phosphatidylcholine-dependent phospholipase D (PC-PLD) is not involved. Our experiments with R59022, a DAG kinase (DAGK) inhibitor, indicate that such an increase may be due to the phosphorylation of DAG derived from phosphatidylinositol (PI) hydrolysis. Our results show that phorbol 12-myristate 13 acetate (PMA) plays a significant role in late DAG formation and that Pcho is released concomitantly, suggesting there is a relationship between the two phospholipase Cs (PLCs) that occurs through a protein kinase C (PKC) translocation from cytosol to the plasma membrane. These findings are confirmed by the use of PKC inhibitors calphostin, H7, and staurosporine. The involvement of membrane phospholipid hydrolysis and the ensuing production of second messengers might explain the vasorelaxant effect of ANF.