The cell signaling pathways that control ventricular cardiomyocyte proliferation during development are poorly understood. Here we show that increasing levels of the ubiquitous Ca(2+) receptor calmodulin (CaM) can regulate cardiomyocyte proliferation in vivo. Targeted overexpression of calmodulin in the heart during embryonic development leads to a 37% or a 79% increase in the number of ventricular myocytes present at embryonic d 17 in mice heterozygous or homozygous for the transgene, respectively. Whereas all homozygous mice die within 10 d after birth, most of the heterozygous mice survive even though they contain 40% more ventricular myocytes relative to the wild-type mice throughout development and into adulthood. The CaM transgene continues to be overexpressed postnatally and, although cell proliferation ceases soon after birth, the elevated levels of CaM lead to an increase in DNA synthesis, which correlates with an increase in the degree of ventricular myocyte polyploidy. Only after proliferation has ceased and polyploidy has become maximal does the continued presence of overexpressed CaM lead to ventricular hypertrophy. However, unlike the case for myocyte number, turning off expression of the CaM transgene results in regression of the hypertrophic response. Together, our results reveal that excess CaM enhances the extent of cell proliferation and DNA synthesis as well as development of hypertrophy of ventricular myocytes in vivo, in a manner consistent with the normal timing of these events during heart development.