The purpose of this study was to examine the role of calcium ions in gallbladder glycoprotein secretion in cultured guinea pig gallbladder explants. The calcium ionophore A23187 showed a threshold of 2 μg per ml medium for stimulation of secretion of [3H]glucosamine-labeled glycoproteins over a 30 min incubation period. The ionophore at 3 and 5 μg per ml medium resulted in a 3- to 4-fold increase in secretion of [3H]glucosamine-labeled glycoproteins. Ionophore-induced stimulation of glycoprotein secretion was abolished by the addition of 0.01 mM verapamil to the medium. To study the effect of changes in extracellular calcium on basal glycoprotein secretion, explants were cultured for 24 hr in media with 0.007, 0.5 or 2.0 mM calcium; no differences in basal glycoprotein secretion were observed. When cultured in medium with 1.0 mM EGTA, basal secretion decreased significantly vs. controls in 0.007 mM total calcium medium. Total [3H]glucosamine incorporation by explants in medium with EGTA was unaltered, however, suggesting that the low level of calcium in the medium was selectively impairing the secretory process. These findings indicate that calcium ions are important in the regulation of gallbladder glycoprotein secretion.