The pre-mRNA encoding the gamma 2 subunit of the gamma-aminobutyric acid Type A receptor is spliced in a tissue-specific manner in mammalian brain resulting in mRNAs containing or lacking a 24 nucleotide exon, gamma 2L and gamma 2S, respectively. The gamma 2S mRNA predominates in pituitary, whereas the gamma 2L predominates in brainstem, spinal cord and cerebellum. In this study, a cell line derived from rat cerebellum that qualitatively reproduces regulated splicing of gamma 2 pre-mRNA was identified and used to dissect cis-regulatory elements. Sequence elements that alter the selection of the 24 nucleotide exon fall into two functional classes-activating elements and inhibitory elements. We identified several inhibitory elements that inhibit splicing of the 24 nucleotide exon in all cell types as well as specific inhibitory elements which repress splicing in non-neuronal cells. Activating elements are localized within conserved intron regions, as judged by a comparison of rat and human gene sequences, and appear to function generally in activating splicing of the 24 nucleotide exon in the cell types tested so far. These results are compatible with a hypothesis in which the mechanism of regulation involves a release from inhibition. Current experiments are aimed toward the development of tools to identify the trans-regulatory components.