Two full-length cDNAs, named CaM1a and CaM1b, encoding the highly conserved calmodulin1 (CaM1) proteins, were isolated from the cDNA library of amphioxus Branchiostoma belcheri tsingtauense.There are only two nucleotide differences between them, producing one amino acid difference between CaM1 a and CaM1b. Comparison of the amino acid sequence of CaM1 reveals that the B. belcheri tsingtauense CaM1a is identical with CaM1 proteins of B. floridae and B. lanceolatum, Drosophila melanogaster CaM,ascidian Halocynthia roretzi CaMA and mollusk Aplysia califoica CaM, and CaM1b differs only at one position (138, Asn to Asp). The phylogenetic analysis indicates that the CaM1 in all three amphioxus species appears to encode the conventional CaM and CaM2 might be derived from gene duplication of CaM1.Southe blot suggests that there are two copies of CaM1 in the genome of B. belcheri tsingtauense. Northe blot and in situ hybridization analysis shows the presence of two CaM1 mRNA transcripts with various expression levels in different adult tissues and embryonic stages in amphioxus B. belcheri tsingtauense. The evolution and diversity of metazoan CaM mRNA transcripts are also discussed.