OBJECTIVE Studies of the acute effects of ethanol on hepatocellular free calcium concentration, [Ca2+]i, and on receptor-operated [Ca2+]i signals have produced conflicting results. The effects of ethanol on basal and receptor-operated [Ca2+]i signals in rat hepatocytes cultured on two different extracellular matrices were examined. METHODS [Ca2+]i was determined by digitized fluorescence microscopy and inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) by high-performance liquid chromatography (HPLC). RESULTS Ethanol induced an increase in [Ca2+]i in hepatocytes cultured on collagen I but not on the more physiological substratum, matrigel. Compared with hepatocytes cultured on matrigel, cells on collagen I exerted less responsiveness and lower amplitude of [Ca2+]i signals to vasopressin or phenylephrine. The effects of ethanol on receptor-operated [Ca2+]i signals were examined in hepatocytes cultured on matrigel. Incubation of hepatocytes with physiologically attainable concentrations of ethanol (20-30 mmol/L) for 30 minutes to 48 hours perturbed epidermal growth factor, phenylephrine, and lower concentration (< or = 1 nmol/L) of vasopressin-induced [Ca2+]i signaling by reducing the amplitude and changing the pattern of [Ca2+]i signals. Alcohol induced-impairment of [Ca2+]i signaling was associated with decreased production of Ins(1,4,5)P3. CONCLUSIONS Alcohol does not itself evoke an increase in [Ca2+]i in hepatocytes cultured on matrigel but perturbs receptor-operated [Ca2+]i signaling. This is associated with and could be caused by impaired generation of Ins(1,4,5)P3.