The zinc-dependent potentiating effect of ethanol (EtOH) on insulin-stimulated DNA synthesis was studied with a focus on the possible site of EtOH action and the ability of other alcohols to elicit similar promitogenic effects. In serum-starved (27 hr) NIH 3T3 fibroblasts, 200-300 mM methanol (MeOH) and 0.1-1.5 mM ethanolamine (Etn), but not 3- to 9-carbon normal alcohols, enhanced the effect of insulin on DNA synthesis to varying extents. The promitogenic effects of EtOH and MeOH, but not that of Etn, required the presence of 15-25 microM zinc. The potentiating effects of Etn were enhanced by 5 mM choline (Cho) and inhibited by 1-3 mM hemicholinium-3 (HC-3), an inhibitor of Cho transporter and Cho kinase. In the presence of 15 microM zinc, 40 mM EtOH, which had no effect on its own, inhibited the potentiating effects of Cho and enhanced the inhibitory effects of HC-3 on synergistic stimulation of DNA synthesis by Etn and insulin. On the other hand, both Cho and HC-3 partially inhibited the promitogenic effect of 80 mM EtOH in the presence of 25 microM zinc. After a 10-min incubation, EtOH decreased the amount of cell-associated [(14)C]Cho in the absence but not in the presence of HC-3. After a 40-min incubation, Cho (5 mM) partially inhibited the cellular uptake as well as the metabolism of [(14)C]Etn. Whereas after the 40-min incubation 80 mM EtOH had no effects on Etn metabolism, in the absence of Cho it decreased the amount of cell-associated [(14)C]Etn. However, EtOH had no detectable effects on cell association of [(14)C]Etn after the 10-min incubation. The results suggest that in NIH 3T3 fibroblasts EtOH is a remarkably specific promitogen, and that it may act via a cell membrane site(s), also regulated by Cho (agonist) and HC-3 (antagonist), which can influence membrane binding and the promitogenic activity of Etn.