In the rat, variation in alcohol and benzodiazepine sensitivity has been correlated with an inherited variant of the GABA(A)alpha6 receptor. Our goal was to identify polymorphisms in the human GABA(A)alpha6 receptor gene and determine whether a variant of the receptor is associated with alcoholism. The GABA(A)alpha6 receptor gene coding region was screened in 80 unrelated patients with alcoholism using single strand conformational polymorphism analysis. For rapid genotyping, a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay was developed. A relatively abundant amino acid substitution and three synonymous DNA substitutions were detected. The synonymous variants, 35A>G, 665A>G, and 1031G>C had rare-allele frequencies of 0.25, 0.02, and 0.47, respectively. The Pro385Ser substitution is located in the second intracellular domain of the receptor adjacent to a putative phosphorylation site. Pro385Ser has rarer allele frequencies of 3.3% and 4.8% in 196 Finnish alcoholic patients and 189 controls, respectively (P = NS). A naturally occurring non-conservative Pro385Ser was detected in the GABA(A)alpha6 receptor. The variant is not associated with alcoholism.