The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.