Using radioimmunoassay, the distribution of radioimmunoassayable neurotensin (R-NT) has been determined in acid/acetone extracts of tissues from week-old and adult rats. Whereas only 10% of the R-NT in week-old rats was found in the head, 90% was found in extracts of the body, and 85% was present in the intestine. The distribution of R-NT in the central nervous system of adult male rats was as follows: hypothalamus (35%), brain stem (35%), cerebral cortex (17%), thalamus (11%), cerebellum (1%), and pituitary gland (1%). Concentrations of R-NT were highest in the hypothalamus (60 pmol/g) and lowest in the cerebellum (0.8 pmol/g). More than 10 times as much R-NT was found in extracts of adult rat small intestine than in brain. The concentration of R-NT was highest in the jejuno-ileal section of the intestine (approximately 50 pmol/g) but it was also detected in the esophogus, stomach, duodenum, and large intestine (approximately 1 to 8 pmol/g) than the remaining muscle layer (approximately 40 pmol/g). Rat jejuno-ileal R-NT co-chromatographed with neurotensin on Sephadex G-25, displayed equal immunological potency with the antisera utilized, and was destroyed by enzymes known to cleave neurotensin. High concentrations (approximately 400 pmol/g) of an R-NT of smaller molecular weight than neurotensin were found in rat stomach. This peptide reacted most strongly with a COOH-terminal-directed antiserum and appeared to be four to five amino acids in length; it might possibly be a breakdown product of neurotensin or a related peptide with biological activity. Acid/acetone extracts of rat and bovine plasma were found to contain an R-NT which displayed the properties of neurotensin (approximately 50 fmol/ml) as well as a substance which behaved similarly to the small molecular weight stomach R-NT (approximately 1000 fmol/ml).