The distribution of binding sites for substance P labeled with [125I]-Bolton-Hunter-reagent was studied in a mixed cell culture preparation from newborn guinea-pig atria and interatrial septum. A relatively small subpopulation of intracardiac neurons expressed substance P binding sites. These neurons exhibited a range of densities of labeling and could be heavily, moderately or lightly labeled with autoradiographic grains. In most cases, the autoradiographic grains were restricted to the neuronal cell body and more proximal regions of the neurites in culture. Intracardiac neurons expressing substance P binding sites were seen in close association with unlabeled neurons. The density of labeling and the distribution of autoradiographic grains over individual intracardiac neurons did not appear to be related to whether they were mono- or binucleate or their associated cell types. The possibility that the substance P binding sites demonstrated here represent functional receptors on intracardiac neurons and their potential role in the heart is discussed.