The troponin C (TnC) superfamily genes generally possess five introns, and the positions where they are inserted are well conserved except for the fourth intron. Based on a structural comparison of TnC genes, we proposed that the common ancestor of TnC or TnC superfamily genes had no intron corresponding to the modern fourth intron, and therefore members of the superfamily have gained the fourth intron independently within each lineage. Here, we cloned calmodulin (CaM, one of the members of the TnC superfamily) cDNAs from two lower marine nonvertebrates, the sea anemone, Metridium senile, belonging to the Cnidaria, and the sponge, Halichondria okadai, belonging to the Porifera, and also determined their genomic organization. Chordate CaM genes generally possess five introns, but neither sea anemone nor sponge CaM has anything corresponding to the fourth intron of chordate CaMs, suggesting that the early metazoan CaM must have had only four introns. The modern fourth intron of chordate CaMs was acquired within the chordate lineage after nonvertebrate/chordate divergence. This notion concurs with our proposal explaining the evolution of the TnC superfamily genes.