The three bona fide genes coding for calmodulin CALM1, CALM2, and CALM3, were assigned to human chromosomes 14, 2, and 19, respectively, by polymerase chain reaction-based amplification of calmodulin gene-specific sequences using DNA from human-hamster cell hybrids as template. Employing calmodulin gene-specific DNA probes of mainly intronic or flanking parts of the individual genes, regional sublocalization was performed by in situ hybridization on metaphase spreads of human lymphocytes. The three calmodulin genes map to chromosomes 14q24-q31, 2p21.1-p21.3, and 19q13.2-q13.3, respectively. These results represent the first complete chromosomal localization study on a mammalian calmodulin gene family and indicate that these structurally closely related genes were most likely dispersed throughout the genome concomitantly with their generation from an ancestral precursor gene.