Mammalian cardiac myocytes express multiple gap junction channel proteins or connexins. Expression patterns of the avian homologues of the mammalian cardiac connexins change during cardiac morphogenesis in association with changes in the electrophysiologic properties of intercellular junctions in chick cardiac myocytes. To determine whether expression of cardiac connexins is developmentally regulated in humans, we characterized connexin mRNA and protein content and distribution in hearts of 11 human fetuses (74 to 122 d gestational age), seven children (0.5 mo to 3 y of age), and two adults. Northern blot analysis identified transcripts of connexin40 (Cx40), connexin43 (Cx43), and connexin45 (Cx45) genes in all hearts analyzed. Cx40 mRNA was approximately 5-fold more abundant in samples from fetal hearts than in hearts of children or adults. However, fetal samples used for RNA extraction included atrial as well as ventricular myocardium, whereas samples from children and adults were exclusively ventricular. Northern analysis of adult human right atrial appendages revealed abundant Cx40 mRNA, thus suggesting that the greater amount of Cx40 signal seen on Northern blots from fetal hearts could have been attributable to atrial contributions. Neither Cx43 nor Cx45 mRNA varied significantly in amount in samples from the different developmental stages analyzed. Immunofluorescence identified abundant Cx43 in the known distribution of gap junctions in myocytes in sections of all hearts. Cx45 staining was inconspicuous in fetal hearts but was readily apparent in cardiac myocytes in hearts of older subjects. In contrast, Cx40 staining in the ventricle was confined to mural coronary arteries, apparently in endothelial cells, whereas in the atrium Cx40 staining at myocyte junctions was abundant. Junctions in hypertrophied myocardium of children with congenital or acquired heart disease appeared more prominent and complex than in normal tissues. Thus, the human heart contains multiple connexins, but their distribution and developmental patterns differ from each other and from those in other species. Cx45 expression is not restricted to the fetal human heart. Cx40 expression in ventricles occurs selectively in coronary arteries.