Recent reports have described modulation of atrial natriuretic factor (ANF) secretion by atrial stretch, increased Na+ concentration, and a variety of hormones and neurotransmitters. One problem in the study of ANF secretion has been the isolation of stimulatory effects from interference due to rhythmic myocardial contraction. To avoid this problem, rat atria were dispersed, the myocytes were suspended in a polyacrylamide gel matrix, and the resulting cell column was perifused with a physiological buffer. The secretion of ANF immunoactivity was markedly stimulated by increases in extracellular osmolality, regardless of the solute (NaCl, KCl, or glucose). This effect did not require extracellular Ca2+, nor could it be mimicked by depolarizing concentrations of K+ in an isotonic medium. Functional viability of this model was demonstrated by significant dose-related increases in ANF release in response to as little as 1 nM epinephrine. The dissociation of ANF secretion from depolarization-induced changes in Ca2+ flux is unusual and may represent an adaptation to the dual roles of the atrial myocyte, contraction and secretion.