Adrenocorticotropin (ACTH) was measured in the cerebrospinal fluid (CSF) of male rats implanted with a push-pull cannula in the 3rd ventricle (ICV) and perfused for 3-6 hr. In 5 conscious sham-lesioned controls studied one week after cannulation, CSF ACTH displayed a pulsatile circhoral pattern with a mean concentration of 10.3 +/- 1.1 pg ACTH/15 min, and a peak/trough ratio of about 2. This pattern was not affected by 2 min of ether vapor inhalation or by pentobarbital anesthesia. The high mortality rate following ICV push-pull cannulation in rats hypophysectomized for 2 weeks led us to perform the ICV perfusion just after cannulation in 5 hypophysectomized rats when they were still under pentobarbital anesthesia. Even so the levels and pulsatile pattern of ACTH release into the CSF were unchanged despite the complete disappearance of any detectable amount of ACTH from the plasma. In two of the latter specimens perfused again one week later but without anesthesia, CSF ACTH levels still were indistinguishable from controls. It can thus be concluded that the ACTH detected in the 3rd ventricle CSF is of neuronal origin.