The structural organization of the entire chicken calmodulin (CaM) gene was determined by analysis of overlapping genomic clones obtained from Charon 4A and cosmid DNA libraries. These clones together span 39 kilobases of chicken genomic DNA. The CaM gene is 12 kilobases long and contains 8 exons interrupted by introns of highly variable size. The first intron (A) is only 19 base pairs (bp) long and divides the 5' untranslated region. Intron B separates the ATG from the first nucleotide of the triplet which encodes the NH2-terminal amino acid of CaM (Ala) whereas intron C separates the triplets encoding amino acids 10-11. Introns D, F, and G interrupt the Ca2+ binding subdomains II, III, and IV of CaM whereas intron E is localized in the linker region between the highly homologous NH2- and COOH-terminal halves of the protein. Primer extension studies using chicken brain poly(A+) mRNA and a fragment from the 5' untranslated region of a CaM cDNA identified the presumptive transcription initiation site (cap site) of the mRNA to be 103 bp 5' from the initiation condon ATG. A consensus sequence TATTTAA was localized 29 bp 5' from this cap site while a CCAAT sequence was located further 5' at position -58 bp. The structure of the CaM gene is strikingly similar to genes that encode other Ca2+ binding proteins from sea urchin, chicken, rat, and quail. These data suggest a conservation of genome organization related to the calcium binding and regulatory domains of Ca2+ binding proteins.