Calmodulin (CaM) levels are developmentally regulated in the mouse heart. During late gestational and early postnatal stages, CaM levels decline several-fold in close temporal association with the declining population of proliferating cardiomyocytes. This correlation suggests that CaM may influence cardiomyocyte cell cycle activity, particularly since CaM is implicated in cell cycle control in several eukaryotic nonmuscle cells. To test this possibility, nucleotides -500 to 77 of the human atrial natriuretic factor gene were linked to a chicken CaM minigene to establish two pedigrees of transgenic mice that express 3- to 5-fold increased levels of CaM in cardiomyocytes. Developmental overexpression of CaM in mouse cardiomyocytes produced a markedly exaggerated cardiac growth response, characterized by the presence of cardiomyocyte hypertrophy in regions demonstrated to overexpress CaM and by cardiomyocyte hyperplasia, apparent at early developmental stages. Early postnatal suppression of fusion gene expression in the cardiac ventricles correlated with regression of the ventricular growth response in transgenic relative to nontransgenic mice between 3 days and 6-10 weeks of age, but was not apparent in the cardiac atria, where levels of CaM remained constitutively elevated until advanced stages. To test the possibility that increased cytosolic Ca2+ buffering contributes to the growth response induced by CaM over-expression, two additional lines of transgenic mice were generated using the same human atrial natriuretic factor promoter to target expression of a CaM mutant (amino acids 75-82 deleted) in cardiomyocytes. This mutant has previously been shown to bind Ca2+ with kinetic properties similar to those of wild-type CaM, but was unable to activate several CaM-dependent target enzymes in vitro. Despite high level expression of the CaM mutant, no growth response was apparent in the hearts of transgenic relative to those of nontransgenic mice, suggesting that increased Ca2+ buffering is unlikely to contribute to the growth response induced by CaM overexpression. Taken together, these findings reveal that cardiomyocyte growth regulation is specifically influenced by CaM concentrations in transgenic mice.