Although recent evidence suggests the presence of the 15-kilodalton (K) mol wt (Mr) precursor of atrial natriuretic factor (ANF) and pro- ANF mRNA in the thymus of human and rat neonates, the cellular origin of the peptides in the tissues remains to be elucidated. We report here that in adult male rats, the 15K Mr presumptive precursor for ANF and a smaller 3K Mr, N-terminal truncated congener of the peptide, ANF-(5-28), are localized in a small population of thymic macrophages. The production of ANF in the thymus was further confirmed by demonstrating the presence of a single band of pro-ANF mRNA signal of approximately 0.8 kilobases from the tissue extract, corresponding to that in the heart. To examine the distribution of pro- ANF mRNA at a cellular level, colorimetric in situ hybridization with digoxigenin-labeled 30-mer oligonucleotides complementry to the first 10-amino acid sequence of ANF-(l-28) was employed. Positive staining was found in thymic cells localized mainly in subcapsular areas, with diffuse staining of positive cells in both the cortex and medulla mainly concentrated around the cortico-medullary junction of the tissue. The identity of the ANF-positive cells was further investigated using a double staining technique to costain immunoreactive (ir) ANF and the macrophage markers EDI and S22 or the T-cell marker OX-19 in both thymic sections and in monolayer cultures of thymic cells. In the latter, approximately 17% of the adherent cells stained positive for irANF after 48 h in culture. Of these cells, more than 95% also stained positive for the macrophage marker EDI, whereas approximately 36% of the EDl-positive cells were colocalized with irANF. In contrast, no irANF- positive cells were colocalised with the pan-T-cell marker OX-19. In tissue sections, irANF was found in cells distributed predominantly around the cortico-medullary junctions and subcapsular areas, with diffuse staining of the cells in the medullary and cortical regions. Sephadex G-50 gel chromatographic profiles of thymic extracts revealed a major peak of immunoreactivity consistent with 15K Mr and a smaller peak that coeluted with rat ANF-(l-28) of 3K Mr. HPLC analysis of the 3K Mr species showed a single peak of immunoreactivity, which eluted with a retention time identical to that of synthetic rat ANF-(5- 28). Thus, our findings confirm that the thymus is an organ of ANF synthesis and demonstrate, in addition to the 15K Mr prescursor of ANF, the production of an N-terminal truncated form of the peptide, ANF-(5-28), by a subpopulation of macrophages in thymic tissues of the adult rat.