Studies were performed to evaluate the contribution of intracellular Ca2+ to gallbladder smooth muscle contraction under acetylcholine (ACh) or potassium stimulation. Gallbladder smooth muscle strips from adult guinea pigs were placed in tissue baths containing N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered physiological salt solution (PSS) and set to optimal length for contraction (Lo). The results were as follows, 1) A 20-min equilibration in zero Ca2(+)-0.1 mM ethylene glycol-bis( beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) PSS virtually abolished the response to potassium but not to ACh. 2) Substitution of strontium, an inhibitor of intracellular Ca2+ release, for Ca2+ significantly decreased the contractile response to ACh (3 X 10(-5), 10(-4), and 3 X 10(-4) M). Strontium had no effect on the response to 40 and 80 mM potassium. 3) Intracellular Ca2+ depletion significantly decreased gallbladder smooth muscle contraction to ACh (10(-4) M) but had no effect on the response to potassium (80 mM). 4) Ryanodine, a compound that inhibits Ca2+ storage by the sarcoplasmic reticulum, significantly decreased the contractile response to ACh (10(-4) M) but not to potassium (80 mM). These data support the observation that the use of intracellular Ca2+ by gallbladder smooth muscle for contraction is agonist dependent.