Alternative RNA splicing greatly increases proteome diversity and may thereby contribute to tissue-specific functions. We carried out genome-wide quantitative analysis of alternative splicing using a custom Affymetrix microarray to assess the role of the neuronal splicing factor Nova in the brain. We used a stringent algorithm to identify 591 exons that were differentially spliced in the brain relative to immune tissues, and 6.6% of these showed major splicing defects in the neocortex of Nova2^−/− mice. We tested 49 exons with the largest predicted Nova-dependent splicing changes and validated all 49 by RT-PCR. We analyzed the encoded proteins and found that all those with defined brain functions acted in the synapse (34 of 40, including neurotransmitter receptors, cation channels, adhesion and scaffold proteins) or in axon guidance (8 of 40). Moreover, of the 35 proteins with known interaction partners, 74% (26) interact with each other. Validating a large set of Nova RNA targets has led us to identify a multi-tiered network in which Nova regulates the exon content of RNAs encoding proteins that interact in the synapse.