We have raised antisera to two synthetic peptides representing different portions of the human pro-atrial natriuretic factor (ANF) molecule; one antiserum identifies active human ANF, the 28-amino-acid sequence on the C-terminal end of the prohormone [ANF (99-126)], and the other detects ANF (1-16), the first 16-amino-acid sequence at the N-terminal end of the prohormone. With ultrastructural immunocytochemistry we have studied the distribution staining for both peptides within the myocytes in surgically excised human auricular appendages. Most of the endocrine granules stained with equal density for both ANF (1-16) and ANF (99-126). Also, double immuno-staining techniques on the same tissue section showed that both the C-terminal peptide and the N-terminal peptide co-existed within the same endocrine granules. It has been shown that, like other endocrine cells, atrial myocytes secrete their stored peptides by exocytosis of their granules. Therefore, our observations suggest that both the main active hormone, ANF (99-126), and the N-terminal propeptide ANF (1-16) are secreted simultaneously from the cell.