Alternative pre-mRNA splicing is frequently used to expand the protein-coding capacity of genomes, and to regulate gene expression at the post-transcriptional level. It is a significant challenge to decipher the molecular language of tissue-specific splicing because the inherent flexibility of these mechanisms is specified by numerous short sequence motifs distributed in introns and exons. In the present study, we employ the glutamate NMDA (N-methyl-D-aspartate) R1 receptor (GRIN1) transcript as a model system to identify the molecular determinants for a brain region-specific exon silencing mechanism. We identify a set of guanosine-rich motifs that function co-operatively to regulate the CI cassette exon in a manner consistent with its in vivo splicing pattern. Whereas hnRNP (heterogeneous nuclear ribonucleoprotein) A1 mediates silencing of the CI cassette exon in conjunction with the guanosine-rich motifs, hnRNP H functions as an antagonist to silencing. Genome-wide analysis shows that, while this motif pattern is rarely present in human and mouse exons, those exons for which the pattern is conserved are generally found to be skipped exons. The identification of a similar arrangement of guanosine-rich motifs in transcripts of the hnRNP H family of splicing factors has implications for their co-ordinate regulation at the level of splicing.