In bovine adrenal glomerulosa cells, angiotensin-II (All) induced a biphasic increase in 1,2-src-diacylglycerol (DAG), with an initial peak at 10 sec followed by a transient decrease at 30 sec. The second increase was much higher in magnitude than the first peak and reached its maximum after 1 h of stimulation. Such kinetics of DAG formation resemble those with which All stimulates the formation of inositol-l,4,5-trisphosphate. The protein synthesis inhibitor cycloheximide, which prevents hormone-induced de novo phospholipid synthesis in adrenal fasciculata cells, had no effect on the DAG response to AH. The first phase of signal generation of both inositol-1,4,5- trisphosphate and DAG was not affected by incubation in calcium- deficient extracellular medium. However, the second phase of the inositol phosphate response was almost completely inhibited in low calcium medium, while the DAG response was reduced by only one third. Pertussis toxin (150 ng/ml) and the voltage-sensitive calcium channel inhibitors, nifedipine (1 μM) and Ni²⁺ (100 μM), had no effect on the DAG response to AIL The retention of a substantial DAG response to All in low calcium medium, with concomitant diminution of the inositol phosphate response, indicates that a major part of the DAG formed during the sustained phase of hormonal stimulation is derived from sources other than phosphoinositides. The DAGs produced from different phospholipids could have distinctive fatty acid compositions and membrane localizations, which, in turn, could result in the differential activation of protein kinase- C. In this way, the increased complexity of the hormonally induced signalling pathway could allow for a greater diversity of responses in hormone-stimulated target cells. (Endocrinology126: 1001–1008, 1990)