To verify the hypothesis that tissue-specific patterns of gap junctions (GJs) are determined by intrinsic factors within myocytes forming different cardiac tissues, we have compared by quantitative transmission electron microscopy (TEM) the structural features of GJs in adult rat atrial myocytes (AMs) and ventricular myocytes (VMs) in vivo with those in developing GJs in cultured AMs and VMs in vitro. Quantitative TEM data revealed a 3-fold increase in the number of developing GJs per intercalated disk in both AMs and VMs from 6 to 15 days in culture. However, at days 12 and 15, the percentage of GJ length per intercalated disk and mean GJ length were 2-fold higher in VMs than in AMS: Measurements of connexin43 GJs by confocal microscopy confirmed TEM data and demonstrated respectively 2- and 4.5-fold greater mean values of GJ length and area in VMs than in AMS: These differences are attributable to the development of large GJs (>3 micrometer) in VMs, closely resembling those observed in VMs in vivo. Although large GJs in cultured VMs comprised approximately 14% of the total number of GJs, their contribution to total GJ length and area constituted >60% and 85%, respectively. In marked contrast, the number of large GJs in AMs both in vitro and in vivo was <1% from the total number of GJS: These data confirm our hypothesis and provide the first evidence that tissue-specific patterns of GJs in AMs and VMs are determined primarily by intrinsic factors within cardiac myocytes and are developmentally regulated.