Alternative splicing of mRNA precursors allows the synthesis of multiple mRNAs from a single primary transcript, significantly expanding the information content and regulatory possibilities of higher eukaryotic genomes. High-throughput enabling technologies, particularly large-scale sequencing and splicing-sensitive microarrays, are providing unprecedented opportunities to address key questions in this field. The picture emerging from these pioneering studies is that alternative splicing affects most human genes and a significant fraction of the genes in other multicellular organisms, with the potential to greatly influence the evolution of complex genomes. A combinatorial code of regulatory signals and factors can deploy physiologically coherent programs of alternative splicing that are distinct from those regulated at other steps of gene expression. Pre-mRNA splicing and its regulation play important roles in human pathologies, and genome-wide analyses in this area are paving the way for improved diagnostic tools and for the identification of novel and more specific pharmaceutical targets.