DNA sequences encoding two variants of a novel gamma-aminobutyric acidA (GABAA) receptor beta subunit were isolated from an embryonic chicken whole-brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named beta 4 and beta 4', respectively. The predicted mature polypeptides are 72-77% identical to the previously characterized mammalian and chicken beta 1, beta 2, and beta 3 subunits. Analysis of the beta 4-subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5'-donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.