Genes with new functions often evolve by gene duplication. Alternative spli
cing is another means of evolutionary innovation in eukaryotes, which allow
s a single gene to encode functionally diverse proteins. We investigate a c
onnection between these two evolutionary phenomena. For similar to 10% of t
he described cases of substitution alternative splicing, such that either o
ne or another amino acid sequence is included into the protein, evidence of
origin by tandem exon duplication was found. This is a conservative estima
te because alternative exons are typically short and, on many occasions, du
plicates may have diverged beyond recognition. Dating exon duplications thr
ough a combination of the available experimental data on alternative splici
ng in orthologous genes from different species and computational analysis i
ndicates that most of the duplications antedate at least the radiation of m
ammalian orders or even the radiation of vertebrate classes. At present, ta
ndem exon duplication is the only mechanism of evolution of substitution al
ternative splicing that can be specifically demonstrated. Along with gene d
uplication, this could be a major route for generating functional diversity
during evolution of multicellular eukaryotes.