Bacteria produce many types of hemolysin that induce diarrhea by mechanisms that are not completely understood. Aeromonas sobria hemolysin (ASH) is a major virulence factor produced by A. sobria, a human pathogen that causes diarrhea. Since epithelial cells in the intestine are the primary targets of hemolysin, we investigated the effects of ASH on ion transport in human colonic epithelial (Caco-2) cells. ASH increased short-circuit currents (Isc) in a dose-dependent manner, and it also activated a 125I efflux from Caco-2 cells. ASH-induced Isc increases and 125I efflux activations were both suppressed by low Ca2+ levels in the extracellular solution or by pretreatment with the Ca2+ chlelator BAPTA-AM. Intracellular Ca2+ levels were increased by ASH in a biphasic fashion characterized by a rapid sharp increase (peak 1) followed by a sustained low plateau (peak 2). ASH-induced peak 1 was inhibited by pretreatment with pertussis toxin, indicating that Ca2+ was mobilized from intracellular stores, and peak 2 was induced by an influx of extracellular Ca2+. Peak 2 but not peak 1 was related to Cl- secretion. These results indicate that ASH activates Ca2+-dependent Cl- secretion.