Ito cells (fat-storing cells) have been implicated in mechanisms of liver fibrosis, and transforming growth factor-p1 is a key factor that stimulates collagen production by Ito cells. Moreover, Ito cells are reported to possess contractile proteins and to contract with ligands. We recently reported the presence of L-type voltage-operated Ca2+ channels in Kupffer cells. In this study, we examined whether Ito cells contain Ca2+ channels and also evaluated the effect of transforming growth factor-pl on Ca"+ channels. Cytosolic free calcium concentration was measured in individual cultured Ito cells with the fluorescent Ca2+ indicator dye fura-2. Partial replacement of extracellular Na+ with K+ caused an increase in cytosolic free calcium, presumably as a result of transmembrane Ca2 + influx. Basal cytosolic free calcium levels were around 40 to 50 nmol/L in both control and transforming growth factor-pl-treated cells. In transforming growth factorpl-treated cells, cytosolic free calcium increased in response to K+ at values as low as 10 mmolL, whereas untreated cells did not respond. Half-maximal increases in cytosolic free calcium in transforming growth factor-pl-treated cells were observed with 63 2 6 mmolL K+. With 100 mmol/L K+, intracellular free calcium increased around fourfold above basal values in transforming growth factor-pl-treated cells but was only increased about twofold in untreated controls. We conclude that this increase in cytosolic free calcium occurs by way of voltage-operated calcium channels; it did not occur in the absence of extracellular calcium and cannot be explained by Na+/Ca2+ exchange mechanisms. The influx of Ca2+ was only about one third as large on subsequent depolarizations