Autoradiographic studies aimed at identifying target cells in the brain for ACTH-like peptides were performed using (3H-7-Phe)-4-Met(O2),8-D-Lys, 9-Phe-ACTH4--9, a behaviorally potent analog of ACTH4--9. The 3H-peptide was injected into the lateral ventricle of hypophysectomized rats that were sacrificed 5, 30, 60, 180, and 240 min later. Dry-mount autoradiograms of brain showed the highest density of silver grains in the ventricular lumen and choroid plexus. In addition, radioactivity penetrated brain tissue as far as 100 microns from the ventricles, and was distributed predominantly over neuropil. Within 5 min after the injection, an intracellular concentration of radioactivity above background levels was observed in a small proportion of cells near the ventricles in the septum, caudate-putamen, preoptic area, hypothalamus, thalamus, amygdala, and hippocampus. The cellular labeling decreased in intensity at greater distances from the injection site and at longer survival intervals, and was no longer evident 4 hr after the injection. The labeled cells were usually small, dark, and often elongated, suggesting that ACTH peptides may act preferentially upon a morphologically distinct class of cells in the brain.