A previous report has described the presence of t-[35S]-butylbicyclophosphorothionate binding sites and GABA-gated Cl- flux in the human neuroblastoma IMR-32 cell line. We now report the further characterisation of this binding site and, even more important, the identification of the GABAA receptor alpha 3 sub-unit expressed in these cells. Cell membranes prepared from IMR-32 cells were screened by immunoblotting for reactivity with various GABAA receptor alpha subunit-specific antibodies. Of these, only anti-Cys alpha 3 454-467 antibodies recognised specifically and in a dose-dependent manner an immunoreactive band. This M(r) 58,000 immunoreactive species and the N-deglycosylated derivatives were both coincident with the respective homologues found in both calf cerebral cortex membranes and purified receptor preparations. This is the first report of the identification of a specific GABAA receptor subunit expressed in a human cell line, and it therefore provides a convenient model for the study of receptor structure and regulation.