Muscarinic antagonists block gallbladder contraction induced by cholecystokinin in vivo but have little effect on gallbladder muscle strips. This study examined the effect of neural blockade on cholecystokinin-octapeptide-induced contraction of the intact guinea pig gallbladder in vitro using cholecystokinin-octapeptide applied to the gallbladder serosa, the lumen, or both compartments simultaneously. Simultaneous cholecystokinin stimulation of both the lumen and serosa was the most potent stimulus to contraction, and the responses were significantly inhibited by atropine and tetrodotoxin. Cholecystokinin in the gallbladder lumen alone evoked contraction by a dose-dependent mechanism that was entirely blocked by atropine or tetrodotoxin. Serosal application of cholecystokinin was the least potent, resulting in contractile responses and low sensitivity to neural blockers comparable to effects reported in muscle strips. The results suggest that cholecystokinin can cause gallbladder contraction by stimulating muscle receptors, neural receptors, or both, and combined neural and muscular stimulation is the most potent contractile stimulus. © 1990.