Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gapjunctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Con-nexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and con-nexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are colocalised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.