Angiotensin II (AngII), a potent peptide hormone, has a well-characterized acute effect on vascular smooth muscle. Recently however, an interest has developed concerning both short term and long term AngII-mediated effects in cardiac muscle cells. Presently, little is known about the biochemical signaling mechanisms activated in cardiac muscle by AngII. To address this issue, experiments were designed using cultured neonatal rat ventricular myocytes exposed to AngII, and then analyzed with biochemical assays to examine specific molecular effects. As a first step, existing procedures for culturing neonatal rat cardiocytes had to be improved. The goal was to further purify otherwise heterogeneous cultures of dissociated ventricular cells into homogeneous cultures of spontaneously contracting cardiocytes. After several established methods were investigated, the use of {dollar}\gamma{dollar}-irradiation at low doses (3500 rads total dose), 24 hours after initially plating the cells in plastic 1.7 cm culture wells, was developed and thoroughly characterized for use in our culture system. Further methods were developed to help maintain these cultures in the absence of serum for 10 to 14 days. The next major goal was to examine signaling pathways which AngII might stimulate in these serum-free, spontaneously contracting cardiocytes. With the use of ({dollar}\sp3{dollar}H) arachidonic acid and ({dollar}\sp3{dollar}H) inositol to label membrane phospholipids, significant evidence was gathered that showed AngII activates two different phospholipases thus generating multiple phospholipid-derived second messengers. Thin layer chromatography, gas chromatography-mass spectrometry, and enzyme inhibition assays revealed that both phospholipase C and phospholipase A{dollar}\sb2{dollar} were activated by AngII in these cardiac cells. Their activation leads to the generation of several second messengers including inositol polyphosphates, diacylglycerol, and unesterified arachidonic acid. Further, receptor subtype-specific antagonists revealed that these processes are mediated in part by two different AngII receptor subtypes, the nonpeptide Losartan-sensitive AT{dollar}\sb1{dollar} and the peptide CGP 42112A-sensitive AT{dollar}\sb2.{dollar} These results indicate that cultured neonatal rat ventricular myocytes, like many other types of cells, are subject to regulation by AngII. A specific role for the second messenger molecules released following exposure to AngII, or the reason why AngII stimulates multiple signaling pathways in a seemingly coordinated fashion through subtype-specific receptors, remains to be elucidated.