The metabolism of atriopeptin prohormone ANF1-126 was examined with the aid of two separate radioimmunoassays, one detecting the C-terminal atriopeptins and the other detecting a fragment of the prohormone N-terminus. Intact prohormone standards are recognized in both assays, whereas the C-terminal atriopeptins are only detected by the atriopeptin assay. Both atriopeptin and N-terminal fragment immunoreactivities were detected in rat plasma and were simultaneously elevated following intravenous administration of desamino-arginine-vasopressin. Atriopeptin immunoreactivity returned to basal levels within 60 min after desamino-arginine vasopressin administration, whereas the N-terminal fragment immunoreactivity remained elevated for more than 2 hr. Analysis of both acid-boiled and sodium dodecyl sulfate-boiled rat atrial extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed the presence of a single high molecular weight species which reacted to both antisera and which comigrated with atriopeptin prohormone standards. Western blots of plasma from desamino-arginine vasopressin-stimulated rats yielded both the low molecular weight C-terminal atriopeptin and a high molecular weight N-terminal fragment-reactive peak which was smaller than the prohormone standards and which did not possess atriopeptin immunoreactivity. A recombinant 128-amino acid atriopeptin prohormone construct, ANF1-126-Arg-Arg, was used as a model substrate for prohormone metabolism. ANF1-126-Arg-Arg was specifically cleaved followed incubation with thrombin to yield the 98-amino acid N-terminal fragment and the C-terminal atriopeptin, AP28-Arg-Arg. Processing of ANF1-126-Arg-Arg by reperfusion through an isolated heart or by incubation in serum yielded identical metabolites to those generated by incubation with thrombin. No significant metabolism was observed following incubation of the prohormone with rat plasma. We conclude that the rat heart contains the necessary enzyme to cleave both endogenous and exogenous prohormone to atriopeptin and that processing by blood enzymes is not required.