The inhibitory effects of several neuroleptic agents on intestinal secretion were examined in vitro by measuring short-circuit current, net Cl flux, and cyclic nucleotide concentration, In rabbit ileal mu cosa, trifluoperazine (0.2-0.5 mM) did not significantly alter basal transport rates, but partially inhibited responses to the following secretagogues: theophylline, 8-Br-cAMP, VIP, dimethyl-PGE2 and heat-stable Escherichia coli enterotoxin (a cGMP agonist). Trifluoperazine completely inhibited the response to Ca ionophore A23187. In human small intestinal mucosa, trifluoperazine (0.1-0.5 mM) inhibited electrical responses to VIP and theophylline almost completely. The inhibitory action of trifluoperazine was manifest only on serosal addition. Trifluoperazine did not significantly alter cAMP or cGMP concentrations either under basal conditions or in the presence of secretagogues. It also did not diminish the electrical response to luminally added D-glucose. Three other neuroleptics were tested and found to have antisecretory action; the order of potencies were trifluoperazine greater than chlorpromazine greater than haloperidol greater chlorprothixene. Since all four agents are potent inhibitors of calcium-dependent regulator in bovine brain, this ubiquitous protein may also be the target for their antisecretory action in intestine.