Gastric smooth muscle of guinea pigs was used to investigate whether the inhibitory effect of calcium antagonists on tonic contraction was accompanied by inhibition of phospholipase C activity. Tonic contraction and [³H] inositol phosphate (IP) formation in response to acetylcholine were measured after pretreatment with verapamil, nifedipine, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxy-benzoate (TMB-8) or EGTA. Verapamil (10 μM), TMB-8 (10 μM) or EGTA (2 mM) significantly inhibited acetylcholine (1 μM)-stimulated tonic contraction but nifedipine (100 nM) did not. Acetylcholine dose-dependently increased the formation of [³H]IP. This effect was not observed in the presence of 2 mM EGTA. Both verapamil and TMB-8 significantly inhibited [³H]IP formation induced by 10 μM acetylcholine, whereas nifedipine did not. In a subsequent study, we measured phospholipase C activity in gastric muscle cell homogenate and in permeabilized cells to determine whether calcium antagonists could inhibit the activity directly. The calcium antagonists did not change the phospholipase C activity of the cell homogenate or the permeabilized cells. But EGTA decreased phospholipase C activity by 50%. These results suggest that the inhibitory effects of verapamil and TMB-8 on acetylcholine-stimulated tonic contraction may be accompanied by inhibition of phospholipase C activity.