We have investigated the GABA(A) alpha(6) subunit molecular composition in two rat lines selectively bred for high or low ethanol preference and consumption, namely Sardinian alcohol-preferring (sP) and Sardinian non-alcohol-preferring (sNP) rats, which have been bred at the University of Cagliari, Italy, since 1981. A total of 27 sP, 22 sNP and 25 control rats belonging to five other different strains, were studied by direct sequencing and amplification refractory mutation system analysis. Among the sNPs, only one was found to be normal, 11 heterozygotes, and 10 homozygotes for the G-->A substitution in codon 100, the same R100Q point mutation previously described in Alcohol Non Tolerant rats, while no other animal showed any mutated allele. Pharmacological studies have extensively demonstrated that this substitution in the mature peptide changes the benzodiazepine-insensitive receptor to a sensitive one. In order to test the functional significance of this mutation in native cerebellar GABA(A) receptors, selective breeding from Q/R rats was employed to obtain a sufficient number of R/R homozygotes. Xenopus laevis oocytes were then injected with cerebellar synaptosomes extracted from Q/Q, R/Q and R/R sNP rats. Consistently, utilizing the two-electrode voltage-clamp technique, GABA-evoked currents mediated by GABA(A) receptors containing the mutated alpha(6) subunit were potentiated by diazepam with about a two-fold increased potency, as compared to receptors containing the wild-type, benzodiazepine-insensitive alpha(6) subunit. Our data show for the first time that a mutated GABA(A) alpha(6) receptor subunit segregates in a rat line which voluntarily avoids alcohol consumption, and further support a possible involvement of the GABA(A) receptor containing a mutated alpha(6) subunit in the genetic predisposition to alcohol preference.