To explore the role of Ca2+ in tight-junction permeability, the Necturus gallbladder was exposed to varying Ca2+ concentrations and to the Ca2+ ionophore A23187 added to the mucosal side (1.9 X 10(-6) to 6.8 X 10(-5) M). Electrophysiological parameters measured in an Ussing-type chamber were correlated with tight-junction morphology revealed by freeze-fracture electron microscopy. In Ca2+-free bathing media, transepithelial resistance decreases and tight-junctional ultrastructure is fragmented. In 1.8 mM Ca2+ media, A23187 induces an initial drop in transepithelial resistance, followed by an increase in transepithelial resistance to a value 20% above base line. At peak response to A23187, NaCl diffusion potentials decrease. Freeze-fracture replicas reveal that the number of junctional strands increase pari passu with junctional depth. Both physiological and morphological changes were partially reversible. The initial decrease in transepithelial resistance coincided with a persistent hyperpolarization of the mucosal cell membrane potential difference and a decrease in the mucosal-to-serosal cell membrane resistance ratio. Thus A23187 alters both the transcellular and paracellular pathway, resulting in opposing effects on transepithelial resistance.