Rat hepatocytes were studied for [Ca2+]i with Fura-2 at the single cell level using a microfluorometer-imaging system which showed that both the number of cells elevating [Ca2+]i and the magnitude of [Ca2+]i increase were directly dependent upon ethanol concentration between 50 mM and 1 M. Peak [Ca2+]i increases ranged from 27 nM with 50 mM ethanol to 57 nM after 1 M ethanol. Ethanol appeared to initiate calcium release from intracellular stores and caused a dose dependent production of inositol(1,4,5) triphosphate (Ins(1,4,5)P3) in hepatocytes. Low concentrations of ethanol (50-100 mM) did not significantly raise Ins(1,4,5)P3 although 300 mM-1 M increased Ins(1,4,5)P3 comparable to that found with vasopressin (5 nM). In summary, physiologic amounts of ethanol raise [Ca2+]i in rat hepatocytes, although at lower levels (50-100 mM) the changes may or may not be related to an Ins(1,4,5)P3 pathway.