gamma-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABA(A)R, in mammalian sperm. In the present study a second subtype, GABA(B)R, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABA(B)R1 (GABA(B)R1a and GABA(B)R1c) and GABA(B)R2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABA(B)R1a and GABA(B)R1c/lb, respectively, determined by Western blot using polyclonal anti-GABA(B)R1 antibody. By an indirect immunofluorescence technique, GABA(B)R1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABA(B)R.