The present studies were undertaken to determine whether a 24 h rhythm occurs in neurotensin (NT) levels in the small intestine of the rat and if so, whether the rhythm depends upon the 24 h cycles of light or feeding. A total of 145 male rats were sacrificed at 4 h intervals and the levels of neurotensin-like immunoreactivity (NTLI) in the middle 30 cm of small intestine were determined by radioimmunoassay with region specific antisera. There was a significant (P less than 0.05) 24 h rhythm in the levels of NTLI in groups of rats maintained under constant illumination or a 12:12 light:dark cycle and fasted for either 24 h or provided food ad libitum. Levels of NTLI ranged from 50 to 140 pm/g and were highest during the early morning (0400-0800 h) and lowest during the afternoon (1200-1600 h). The NTLI from samples taken at 0400 and 1600 h was subjected to high-performance liquid chromatography. The levels of chromatographically and immunochemically characterized NT were consistent with the levels of NTLI, evidence that the 24 h variation in NTLI most likely reflects changes in the intestinal content of NT and not other substances with similar immunochemical properties.