ABSTRACT: γ-Aminobutyric acid (GABA), which is a major inhibitory neurotransmitter in the brain, is also found in many peripheral nonneuronal tissues, including male reproductive organs. However, the distribution of GABAergic cells in various organs is not known. The GAD67-GFP knock-in mouse is a useful model for studying the distribution and morphology of GABAergic neurons in the brain. We examined the male reproductive organs of GAD67-GFP knock-in mice by fluorescence microscopy and found cells with strong green fluorescent protein (GFP) signal exclusively in the epithelium of the initial segment and proximal caput of the epididymis. The characteristic cell morphology suggested that these were narrow cells. These GFP-positive narrow cells also expressed GAD67 and GABA. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that the predominant glutamic acid decarboxylase (GAD) isoform expressed in the epididymis is GAD67. RT-PCR analysis also revealed that mRNAs encoding the GABAA and GABAB receptor subunits necessary for the assembly of functional receptors are expressed in the epididymis. GABAA receptor subunit mRNAs detected in the proximal epididymis included α2, β1, γ1, and γ3, and both the R1 and R2 subunit mRNAs of GABAB receptors were detected. Immunohistochemical analysis of GABAA receptor subunit proteins revealed that α2, β1, and γ subunits expressed in spermatozoa, whereas we did not detect these GABAA receptor subunits in epithelial cells. GABAB receptors were produced by narrow cells and spermatozoa of GAD67-GFP knock-in and wild-type Jcl:ICR mice. Our data suggest that the GABA system might have important functional roles in narrow cells and on spermatozoa in the lumen.