The mechanism of phospholipase (PLC)-delta activation by G protein-coupled receptor agonists was examined in rabbit gastric smooth muscle. Ca(2+) stimulated an eightfold increase in PLC-delta1 activity in permeabilized muscle cells. Treatment of dispersed or cultured muscle cells with three G(i/o)-coupled receptor agonists (somatostatin, delta-opioid agonist [D-Pen(2),D-Pen(5)]enkephalin, and A(1) agonist cyclopentyl adenosine) caused delayed increase in phosphoinositide (PI) hydrolysis (8- to 10-fold) that was strongly inhibited by overexpression of dominant-negative PLC-delta1(E341R/D343R; 65-76%) or constitutively active RhoA(G14V). The response coincided with capacitative Ca(2+) influx and was not observed in the absence of extracellular Ca(2+), but was partly inhibited by nifedipine (16-30%) and strongly inhibited by SKF-96365, a blocker of store-operated Ca(2+) channels. Treatment of the cells with a G(q/13)-coupled receptor agonist, CCK-8, caused only transient, PLC-beta1-mediated PI hydrolysis. Unlike G(i/o)-coupled receptor agonists, CCK-8 activated RhoA and stimulated RhoA:PLC-delta1 association. Inhibition of RhoA activity with C3 exoenzyme or by overexpression of dominant-negative RhoA(T19N) or Galpha(13) minigene unmasked a delayed increase in PI hydrolysis that was strongly inhibited by coexpression of PLC-delta1(E341R/D343R) or by SKF-96365. Agonist-independent capacitative Ca(2+) influx induced by thapsigargin stimulated PI hydrolysis (8-fold), which was partly inhibited by nifedipine ( approximately 25%) and strongly inhibited by SKF-96365 ( approximately 75%) and in cells expressing PLC-delta1(E341R/D343R). Agonist-independent Ca(2+) release or Ca(2+) influx via voltage-gated Ca(2+) channels stimulated only moderate PI hydrolysis (2- to 3-fold), which was abolished by PLC-delta1 antibody or nifedipine. We conclude that PLC-delta1 is activated by G(i/o)-coupled receptor agonists that do not activate RhoA. The activation is preferentially mediated by Ca(2+) influx via store-operated Ca(2+) channels.