Atrial natriuretic peptide (ANP) is released from the atria and acts to regulate blood volume and pressure. The release of ANP appears to be stimulated by atrial distension, initiated by stretch on the cardiocytes. The purpose of the present study was to develop an assay that would allow for the detection of ANP release from single, isolated cells in the absence of distension. Using the reverse hemolytic plaque assay and antibody raised against human-alpha ANP, the release of ANP was detected from trypsin dissociated rat atrial cells. The specificity of the assay was demonstrated by a 67% reduction in ANP plaque forming cells detected following preabsorption of the anti-sera with rat-alpha ANP. The assay also proved efficient in monitoring changes in ANP secreting cell populations, where an acute treatment with dexamethasone resulted in a doubling of the percentage of atrial cardiocytes detected within a 4 hour antibody incubation. Finally, the assay established that about 52% of the dispersed atrial cardiocytes release ANP. The establishment of a plaque assay for ANP release should assist in addressing questions concerning what hormones may regulate ANP secretion directly and also allow for the determination of ANP secreting cell population dynamics.