Separate demonstration of immunoreactive (ir) atrial natriuretic factor (ANF) and ir-β-endorphin (β EP) in macrophages of the rat spleen prompted a reexamination of their distribution and cellular localization in addition to their developmental regulation. Double labeling immunohistochemistry was carried out on paraffin-embedded spleen sections of adult male Sprague-Dawley rats. Cells stained positive with antiserum (S118) raised against rat ANF-(99-126) were colocalized in more than 95% of cases with immunofluorescent staining of irβ EP-(1-31) and distributed sparsely throughout the venous sinusoidal regions of the red pulp. In 1- or 5-day monolayer cultures of adherent splenocytes, 11-16% of the cells stained positive for either irANF or irβ EP. Under these conditions, more than 95% of irANF- and irβ EP-positive cells were also fluorescence stained for the histiocyte marker S22 or the rat macrophage marker ED-1. Colorimetric in situ hybridization similarly revealed signals for pro-ANF mRNA in more than 95% of irβ EP-positive adherent cells. Thus, taken together with previous reports, our present findings suggest that in the rat spleen, both ANF andβ EP are produced by the same population of macrophages. However, the tissue levels and processing of the two peptides over the developmental period of the animal differed markedly in several ways. In splenic extracts of 2-day-old neonatal rats, Northern blot analysis and RIA revealed a greater abundance of pro-ANF mRNA signal and an irANF concentration about 6-fold greater than those in 30-day-old animals. In contrast, the irβ EP concentration did not vary significantly over the same period, consistent with the level of POMC mRNA. Sephadex G-50 gel chromatography of splenic extracts from 2-day-old animals revealed predominantly higher mol wt forms of irANF and irβ EP. From days 16-60, a significant proportion of irANF eluted in the same fractions as the mature circulating form, rat ANF-(99-126); the proportion of 3.5-kilodalton irβ EP increased progressively to become the predominant species in adult tissues. Thus, it appears that although ANF andβ EP are coexpressed by the same population of splenic macrophages, they differ markedly during the developmental period with respect to their constitutive regulation.