The highly conserved, intracellular calcium binding protein calmodulin is present in all cells at all times. In addition to this constitutive level, the amount of calmodulin is highly regulated according to the tissue or stage of development. Since there are only a few genes or a single gene for this protein in most species, intricate regulatory elements may be necessary to effect its complex regulation. This report adds new information concerning the gene structure and outlines the developmental and spatial regulation of Drosophila melanogaster calmodulin transcripts. The gene contains five exons, including a 49 bp exon in the 5' untranslated region, and spans over 16 kb. Homologues to this small, 5' noncoding exon have not been found in other calmodulin genes. The combined level of the transcripts is developmentally regulated, and the relative amounts of the two transcript size classes (1.65 kb and 1.9 kb) are differentially regulated during development. Primer extension experiments and RNase protection mapping show that both size classes of Drosophila calmodulin transcripts initiate at the same site but undergo alternative termination within the final exon. The spatial distribution of calmodulin transcripts was examined by in situ hybridization to sections of adults and to developmentally staged whole mount embryos. Calmodulin transcripts are evenly distributed early in embryogenesis. In later stages of embryogenesis, higher levels accumulate in the developing nerve cord and other tissues. Elevated levels of calmodulin transcripts are seen quite distinctly in the adult neural tissues and in the photoreceptor region of the compound eye.