Release of rat urodilatin (rURO) from isolated perfused rat kidneys and neonatal rat kidney cells could be demonstrated by a specific competitive radioimmunoassay (rURO-RIA) using [125I]rURO as the competitive antigen and an antiserum against the hypothetical rURO-N-terminus, Ala-Gly-Pro-Arg, as concluded from the amino acid sequence of the rat prohormone CDD/ANP-1-126. This antiserum did not react with synthetic rCDD/ANP-99-126, brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), or human URO (hURO). rURO could be demonstrated in the urine of the perfused rat kidney after an equilibration period of 20 min. After an initial slight decrease in the second 20 min, rURO production remained at almost the same level during the perfusion time of 100 min. A total of approximately 470 fmol.10 -1.g-1 kidney rURO was produced within 80 min. rURO was also produced by neonatal rat kidney cells kept in serum-free Dulbecco's modified Eagles medium. The production of rURO depended on the cultivation time of the cells. It increased up to 3 days reaching 239 +/- 7.5 fmol.h-1.g-1 protein, afterwards it decreased rapidly. The results obtained indicate that the rat kidney produces a peptide of the type A family of natriuretic peptides, which very likely represents the putative rURO.