The mammalian nervous system is rich in signaling mediated by heterotrimeric (alphabetagamma) G proteins. As an initial step to define the roles that particular gamma subunit types play in signaling, we have begun to clone and characterize those genes that encode gamma subunits enriched within neural tissue. In the present study, we have isolated and characterized the mouse gamma3 subunit gene (Gng3). The gamma3 subunit is expressed abundantly in the brain and at low levels in testes. Gng3 is composed of three exons spanning approximately 1.4 kb. A comparison of Gng3 with the gene structure for five other gamma subtypes indicates that although these proteins are diverse at the amino acid level, their exon-intron boundaries are conserved. Sequence analysis of the 5' flanking region of Gng3 revealed the presence of a novel gene, the gamma3 linked gene (Gng3lg). Gng3 and Gng3lg are organized in a head-to-head fashion with major transcription initiation sites separated by approximately 133 bp. Sequence analysis of a Gng3lg cDNA clone revealed an open reading frame encoding a 410-amino-acid protein of unknown function. Gng3lg transcripts are expressed in a variety of tissues including both brain and testes. Using an interspecific backcross panel, we localized both Gng3 and Gng3lg to the same locus on chromosome 19. The orientation, close proximity, and expression pattern of these two genes raise the distinct possibility that shared regulatory elements are used to control their expression.