The effect of trifluoperazine (TFP) and phencyclidine (PCP) on acetylcholine receptor (AChR) function was studied in rat myotubes differentiated in vitro. While both drugs exerted an inhibitory effect on carbamylcholine (CCh)-induced Na+ or Ca2+ flux (I50 = 5-9 μM), α-bungarotoxin (α-Bgt) binding was not affected. The inhibitory effect of both drugs was independent of CCh concentration. The mutual inhibitory effect of TFP and PCP on Ca2+ influx was analyzed using three alternative models of interaction between the two drugs: competitive, additive and synergistic inhibition models. Our results are in accord with a synergistic interaction between the drugs probably not through desensitization. This synergistic interaction between the drugs provides a biochemical rationale to the phenothiazine contraindication in the treatment of PCP psychosis.