Elsewhere, we have reported the structure of a rat calmodulin gene and two distinct rat calmodulin cDNAs, pRCM1 and pRCM3. Here, I report the cloning and sequencing of the third calmodulin cDNA (pRCM4) and two additional rat calmodulin genes. The original calmodulin gene is named CaM I (pRCM1) and the newly discovered calmodulin genes are named CaM II (pRCM3) and CaM III (pRCM4). CaM II spans about 10 x 10(3) base-pairs and consisted of five exons, while CaM III spans about 7.2 x 10(3) base-pairs and consisted of six exons. One of the introns (intron 3) observed in CaM I and CaM III is lost in CaM II. Otherwise, the intron/exon organization of these genes is exactly the same. In all calmodulin genes, the first intron separates the initiation codon (ATG) from the coding region of the protein. Northern blotting showed that CaM I is transcribed primarily into 1.7 x 10(3) base-pair mRNA in various tissues examined and 4.0 x 10(3) base-pair mRNA mainly in skeletal muscle, CaM II is transcribed into 1.4 x 10(3) base-pair mRNA almost exclusively in brain and CaM III is transcribed predominantly into 2.3 x 10(3) base-pair mRNA and faintly into 1.0 x 10(3) base-pair mRNA mainly in skeletal muscle and brain. DNA sequences in the promoter-regulator regions of these genes are partly homologous but essentially distinct and possess a number of direct repeats, palindromes and feasible stem-loop structures. Together with these, I report here the structures of the third and fourth calmodulin retropseudogenes.