Gastrin can readily be concentrated from 10 to 50 ml of urine with better than 90% recovery using octadecylsilyl (ODS) silica columns (C18 Sep-Pak cartridge) and then measured by radioimmunoassay. Fractionation on Sephadex G50 gel filtration reveals that the apparent immunoreactivity is not due to nonspecific interference in the assay system but does correspond to the two known forms of gastrin, the 17 and 34 amino acid peptides. Renal clearance of gastrin in 5 normal subjects does not appear to differ in the fasted and fed state and ranged from 0.09 to 0.26 ml/min with an average of 0.16 +/- 0.05 (S.D.) ml/min. Urinary gastrin excretion in the overnight fasting state was generally less than 0.005 pmol/hr/kg body weight and fell to lower levels after a 20-hour fast. Increased urinary gastrin output was observed following feeding. Gastrin output in urine in 7 subjects ranged from 6.8 to 10.2 pmol/24 hr with an average of 8.5 +/- 1.5 (S.D.) pmol/24 hr. A single determination of renal gastrin clearance and 24-hour gastrin urinary output appears to be sufficient for the determination of averaged plasma gastrin levels in normal subjects without renal disease. Similar methodology should be applicable to a variety of other peptidal hormones as well.