Two radioimmunoassays for alpha-human atrial natriuretic polypeptide (alpha-hANP) with different specificities were used to study the tissue level and the nature of alpha-hANP-like immunoreactivity in the bovine adrenal gland. A considerable amount of alpha-hANP-like immunoreactivity was detected in the adrenal medulla (90.8 +/- 21.1 and 90.0 +/- 23.1 ng/g with the two radioimmunoassays), while no detectable amount (less than 1.0 ng/g) was present in the cortex. Gel permeation chromatographic analysis showed that ANP in the medulla is composed of two components of alpha-hANP-like immunoreactivity with high and low molecular weights in the approximate ratio of 2:1, eluting at the elution positions of gamma-hANP and alpha-hANP, respectively. Reverse-phase high performance liquid chromatographic analysis revealed that alpha-hANP-like immunoreactivity with a low molecular weight in the medulla consists of two major components, which comigrate with synthetic alpha-hANP(5-28) and alpha-hANP. When cultured bovine adrenal chromaffin cells were incubated in the presence of nicotine (10(-5) M), alpha-hANP-like immunoreactivity was released into the medium concomitantly with catecholamines from chromaffin cells. These findings indicate that a discrete ANP system is present in the adrenal medulla and that ANP is cosecreted with catecholamines from chromaffin cells, suggesting the possible involvement of ANP in the adrenomedullary function.