Neurotrophin-3 (NT-3) is a member of the neurotrophin family of growth factors, best characterized by its survival- and differentiation-inducing effects on developing neurons bearing the trk C receptor tyrosine kinase. Through analysis of NT-3 and trk C gene-targeted mice we have identified NT-3 as critically regulating cardiac septation, valvulogenesis, and conotruncal formation. Although these defects could reflect cardiac neural crest dysfunction, the expression of NT-3 and trk C by cardiac myocytes prior to neural crest migration prompted analysis of cell-autonomous actions of NT-3 on cardiac myocytes. Retroviral-mediated overexpression of truncated trk C receptor lacking kinase activity was used to inhibit activation of trk C by endogenous NT-3, during early heart development in ovo. During the first week of chicken development, expression of truncated trk C reduced myocyte clone size by more than 60% of control clones. Direct mitogenic actions of NT-3 on embryonic cardiac myocytes were demonstrated by analysis of BrdU incorporation or PCNA immunoreactivity in control and truncated trk C-expressing clones. Inhibition of trk C signaling reduced cardiac myocyte proliferation during the first week of development, but had no effect at later times. These studies demonstrate that endogenous NT-3:trk C signaling regulates cardiac myocyte proliferation during cardiac looping and the establishment of ventricular trabeculation but that myocyte proliferation becomes NT-3 independent during the second week of embryogenesis.