Low concentrations of angiogenin activate the inositol-specific phospholipase C of cultured pulmonary artery, umbilical vein, and capillary endothelial cells, promoting a transient increase in the intracellular levels of 1,2-diacylglycerol and inositol trisphosphate. The response is strongly dose dependent with a maximum in the ng/ml concentration range and, for some cell lines, a marked decrease at concentrations greater than 1 ng/ml; e.g., arterial endothelial cells respond weakly to angiogenin concentrations comparable to that in normal human plasma (approximately equal to 400 ng/ml). Chemical modification of the active site of angiogenin or inhibition with placental ribonuclease inhibitor abolishes its activation of endothelial cell phospholipase C; this correlates with the concomitant loss of both intrinsic ribonucleolytic and angiogenic activity. The response to low concentrations of angiogenin is consistent with its potency of inducing vascularization in classical angiogenesis assays. In vivo, endothelial cells are exposed to concentrations of angiogenin higher than that required to elicit a cellular response; it seems likely, therefore, that expression of a surface receptor or some other process must be rate limiting in the cellular response.