Connexin 43 (Cx43) knockout mice and transgenic mice (CMV43) overexpressing the Cx43 gap junction gene exhibit heart defects involving the conotruncus and right ventricle. Based on the heart phenotype and Cx43 gene and transgene expression pattern, we previously proposed that the heart defects may reflect a role for gap junctions in the modulation of cardiac neural crest development. To further elucidate the mechanism by which these heart defects may arise, fetal heart structure and function in these transgenic and knockout mice were examined by magnetic resonance microscopy and Doppler echocardiography. Magnetic resonance microscopy of E14.5 fetuses revealed an enlargement of the right ventricular chamber in the heterozygous Cx43 knockout and CMV43 transgenic mice. This was accompanied by thinning of the chamber wall. In the homozygous Cx43 knockout mouse, heart malformation was also restricted to the right ventricle. This was generally characterized by two pouches at the base of the pulmonary outflow tract, but occasionally hearts with a single pouch were found. Magnetic resonance microscopy showed in some of the CMV43 and Cx43 knockout mice an attenuation of the ductus arteriosus, a phenotype which may be indicative of outflow tract obstruction. This was confirmed by the in utero Doppler echocardiography, which showed increased outflow velocity in E12.5 to 14.5 CMV43 and Cx43 knockout fetuses. In some of these fetuses, Doppler analysis also revealed arrhythmia and absence of isovolemic contraction time. Further examination of these hearts by histology and immunohistochemistry showed abnormal myocardial development in the conotruncus. Particularly interesting was the presence of abundant subendocardial fibrous tissue expressing smooth muscle actin. In the developing heart, such mesenchyme in the outflow tract is usually considered neural crest-derived tissue. Together, these results confirm the importance of Cx43 gene dosage in conotruncal heart development and suggest that this likely involves a role for Cx43 gap junctions in cardiac crest development. In future studies, these transgenic mice may serve as valuable animal models for further studying the role of gap junctions and cardiac crest cells in conotruncal heart development.