Gap junctions have been demonstrated ultrastructurally in cardiac regions where connexin40 (Cx40) and connexin43 (Cx43) protein could not be detected immunohistochemically. We investigated therefore the distribution of their mRNAs with more sensitive techniques. In situ hybridizations with Cx40 and Cx43 cRNA probes were performed on sections of rat hearts from 9 embryonic days (ED 9) to adults. From ED 13, Cx40 and Cx43 mRNA are detectable in atria and ventricles, but not in their flanking myocardium (inflow tract, atrioventricular canal and outflow tract). Even though Cx40 and Cx43 mRNA eventually become expressed in the inflow tract, they remain undetectable in the sinoatrial node, the atrioventricular canal (including atrioventricular node) and outflow tract. Expression of Cx40 is maximal in the fetal period and declines towards birth. Cx40 expression in the left and right ventricles evolves independently, its mRNA disappearing 4 days earlier from the right than from the left ventricle, and earlier from the free wall than from the trabeculations. Expression of Cx43 mRNA increases during development and changes postnatally from uniform to punctate. Prenatally, Cx43 mRNA was strongest in the subepicardial layer of the ventricular free wall. Nevertheless, we did not detect protein in this layer. Cardiac regions without detectable Cx40 or Cx43 mRNA either have extremely low levels of expression or express a different connexin. The temporally separate disappearance of Cx40 mRNA from the fetal ventricles implies that left and right ventricles mature independently with respect to gap-junctional communication. The division of the developing heart in compartments where Cx40 and Cx43 mRNA can and cannot be detected, implies pretranslationally regulated gene expression. The postnatally observed subcellular redistribution of Cx43 mRNA coincides with a reported increase in protein expression.