Tissue- and stage-specific alternative splicing events are widespread in mammals, yet the factors and mechanisms that direct these important posttranscriptional events are poorly understood. In this study, we focus on the 24-nt exon of the GABA(A) receptor gamma2 pre-mRNA, which is subject to neuron-specific and developmental splicing regulation in the rat cerebellum. Here we show biochemical evidence for a mechanism that directs the selective repression of the neuron-specific exon in non-neuronal splicing extracts derived from HeLa cells. Key evidence includes the discovery that the pathway of gamma2 pre-mRNA splicing switches from exon skipping to exon selection in splicing reactions with a short RNA competitor containing the 3' splice site region upstream of the 24-nt exon. In this assay, exon selection results from the coordinate activation of both flanking introns. A detailed dissection of this pre-mRNA region shows that it contains four repressor sites clustered around the branch site and extending into the 24-nt exon. These repressor sites are pyrimidine rich and bind avidly to the polypyrimidine tract binding protein (PTB) in HeLa nuclear extracts as determined by UV crosslinking/competition assays. Repression of the exon selection pathway is closely associated with the appearance of a specific RNA-protein complex, indicative of an inhibitor complex, that assembles on the repressor array. Upon the switch to the exon selection pathway, a substantial decrease in the inhibitor complex and a reciprocal increase in spliceosome complex A is observed. Excess recombinant PTB squelches the splicing switch and reestablishes exon skipping as the predominant splicing pathway. Extracts prepared from rat brain nuclei show reduced levels of conventional PTB compared to other splicing factors. Nonetheless, the rat brain nuclear extracts contain an activity that assembles an analogous inhibitor complex efficiently. We report a 59-kDa protein, p59, which has an electrophoretic mobility distinct from HeLa and rat kidney PTB, and which behaves in RNA binding assays as if it is the PTB counterpart in rat brain. Evidence that rat brain p59 is structurally related to PTB stems from western blot and immunoprecipitation analysis with a monoclonal antibody specific for the hnRNP I isoform of PTB. A model describing how the repressor array directs coordinate splicing regulation of flanking introns in the context of overlapping positive regulatory elements is discussed. The sequence, (5') UUCUCU (3'), in a pyrimidine context is associated with one class of intron splicing repressor sites that binds PTB in a variety of pre-mRNAs that are regulated by tissue-specific programs.