Expression of the gene encoding atrial natriuretic factor (ANF) during mouse cardiac embryogenesis was studied using in situ hybridization to histologic sections. ANF mRNA was first detected in a subpopulation of myocardial cells at day 8 of embryogenesis. During day 9, abundant hybridization of the ANF probe to the atrium and the primitive ventricle was found. At day 14, strong labeling of cells in both atria and the trabeculated regions of the left ventricle was seen, whereas fewer cells in the right ventricle expressed the ANF gene. The developmental regulation of ANF gene expression in atrial and ventricular cells suggests an important role for this peptide hormone in the embryo. Localized expression of the ANF gene provides a marker for discerning subpopulations of cardiac cells during embryogenesis. Identification of embryonic ventricular ANF mRNA transcripts may have implications with respect to pathologic reexpression of the ANF gene in adult ventricles.