In the present study acute effects of ethanol on [3H]-acetylcholine ([3H]-ACh) release induced by activation of strychnine-sensitive glycine receptors in superfused slices of rat caudatoputamen were investigated. The glycine-evoked [3H]-ACh release (Ig EC50 = -4.10, CI95 = [-4.14, -4.05]) was inhibited by strychnine in a competitive manner (pA2 = 6.86, CI95 = [6.61, 7.08]). Release of [3H]-ACh could also be induced by L-serine. L-serine was less potent than glycine (Ig EC50 = -2.61, CI95 = [-2.69, -2.52]). Both glycine and L-serine showed similar maximum effects (Emax(glycine) = 1.34, CI95 = [1.24, 1.45]; Emax(L-serine) = 1.19, CI95 = [1.09, 1.32]). Ethanol at concentrations of 2%/1000 (= 34 mM) and 4%/1000 (= 68 mM) inhibited glycine-evoked [3H]-ACh release in a manner like the competitive antagonist strychnine, however with lower potency. The pA2 of ethanol was 1.19, CI95 = [0.85, 1.41], at 2%/1000 [v/v] and 1.51, CI95 = [1.19, 1.78] at 4%/1000 ethanol. Similar to its action on glycine-evoked [3H]-ACh release, ethanol at 4%/1000 [v/v] also inhibited L-serine-evoked transmitter release in a competitive-like fashion (pA2 = 0.83, CI95 = [-0.15, 1.18]). We conclude, that strychnine-sensitive glycine receptors, mediating [3H]-ACh release in the rat caudatoputamen, might represent a new site of action of ethanol.