Ethanol (1-200 mM), a potent depressor of respiration and motor activity, potentiated the inhibitory Cl- current activated by glycine in 80% of the cultured mouse spinal (n = 236) neurons studied. Ethanol (100 mM) had no effect on the gamma-aminobutyric acidA current and slightly inhibited the N-methyl-D-aspartate current in these neurons. Ethanol increased the affinity of the receptors to glycine without changing the maximal amplitude of the glycine current. The EC50 was reduced from 54 +/- 3 microM in the absence of ethanol to 38 +/- 5 microM in the presence of ethanol. Activation of GTP binding proteins in the neurons with intracellular guanosine-5'-0-(2-thiotriiphosphate) (0.5 mM) enhanced the effect of ethanol, and application of a similar concentration of guanosine 5'-0-(2-thiodiphosphate had an inhibitory effect upon the current potentiation. The potentiating effect of ethanol persisted after culturing the neurons with pertussis toxin, but not with cholera toxin, an irreversible activator of Gs. Activation of cyclic AMP-dependent protein kinase by cyclic AMP and Sp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt, but not of protein kinase C and protein kinase G, potentiated the glycine current. The effect of Sp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt, but not of ethanol, was inhibited completely by the protein kinase A peptide inhibitor. These results suggest that ethanol potentiates the glycine activated Cl- current by modifying a signal transduction step other than protein kinase A.