Early studies indicated that the BaCl2-induced contractions in the guinea pig ileum longitudinal muscle strip (GPI-LMS) were, in part, neuronal in origin. However, recent studies have suggested that BaCl2-induced contractions were produced by an action directly on the smooth muscle membrane. The purpose of this study was to investigate the mechanism of the BaCl2 contractions in the GPI-LMS. Botulinum toxin (5 × 105 MLD/mL), which blocks the electrically induced release of acetylcholine (ACh), hemicholinium-3 (HC-3; 110 μM), which blocks ACh synthesis, tetrodotoxin (TTX; 60 nM), which blocks Na+ channels, black widow spider venom, which depletes the presynaptic neuron of neurotransmitter, and atropine (2.9 μM), a potent muscarinic antagonist, had no effect on the BaCl2 contractions. Desensitization of the GPI-LMS to substance P did not affect the BaCl2 contraction. In Ca2+-free buffer the BaCl2 dose–response curve was shifted to the right. In Ca2+-free solution the time to 50% inhibition of the contractile response to ACh (73 nM) and BaCl2 (1.16 mM) was 3.7 and 125 min, respectively. The D 600 IC50 for ACh and BaCl2 contractions was 220 and 130 nM, respectively. In Ca2+-free buffer either EGTA (0.53 mM) or D 600 (1 μM) were potent inhibitors of BaCl2 contractions. These results suggest that in the GPI-LMS the BaCl2 response is not mediated by a release of ACh (or substance P) because inhibitors of ACh release, synthesis, and receptors do not affect the responses. Also, the BaCl2 contraction is not due to activation of Na+ channels because TTX is without effect. The BaCl2-induced contraction appears to be mainly due to the movement of membrane bound Ca2+ through D 600 sensitive Ca2+ channels with extracellular Ca2+ and possible passage of Ba2+ ions intracellularly playing relatively minor roles.