The evolutionary history of the intracellular calcium-binding protein superfamily is well documented. The members of this gene family are all believed to be derived from a common ancestor, which, itself, was the product of two successive gene duplications. In this study, we have compared and analyzed the structures of the recently described genes coding for these proteins. We propose a series of evolutionary events, which include exon shuffling and intron insertion, that could account for the evolutionary origin of all the members of this superfamily. According to this hypothesis, the ancestral gene, a product of two successive duplications, consisted of at least four exons. Each exon coding for a peptide (a calcium-binding domain) was separated by an intron that had mediated the duplication. Each distinct lineage evolved from this ancestor by genomic rearrangement, with insertion of introns being a prominent feature.