We investigated the release of [3H]arachidonic acid ([3H]AA) and its relationship to the formation of [3H]inositol trisphosphate ([3H]IP3) elicited by substance P (SP) in prelabeled Chinese hamster ovary cells stably expressing the SP receptor. Activation of the SP receptor resulted in a concentration- and time-dependent stimulation of [3H]AA release. Half-maximal release was obtained at 10(-9) M, comparable to that for [3H]IP3 formation reported previously, and the maximal release effected by 0.1 microM SP was 8 to 10-fold above the basal value. Both the [3H]AA release and the [3H]-IP3 accumulation stimulated in the cells by 0.1 microM SP were concentration-dependently blocked with the specific SP receptor antagonist CP-96,345, with IC50 values of 2.5 and 0.4 microM, respectively. The time course of [3H]AA release showed a biphasic pattern: an initial rapid release essentially independent of Ca2+, followed by a sustained release markedly suppressed by removal of extracellular Ca2+ or chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxyethane)-N,N,N',N'-tetraacetic acid (BAPTA). While pretreatment with pertussis toxin (200 ng/mL, 6 hr) did not block [3H]IP3 formation, it did reduce [3H]AA release by 50% at 1 and 10 min after SP stimulation. Treatment of the cells with a phorbol ester, a protein kinase C activator, augmented the SP-stimulated [H]AA release, and sphingosine, a protein kinase C inhibitor, reversed the phorbol ester-potentiated [3H]AA release, but not the release stimulated by SP alone, suggesting a synergistic effect of protein kinase C on SP-stimulated AA release. These results demonstrate that SP, acting at the SP receptor, stimulates [3H]AA release via mechanisms that are (1) mediated by a pertussis toxin-sensitive G-protein, (2) dependent on extracellular Ca2+, and (3) enhanced by activation of protein kinase C.