Abstract The possibility was explored that certain central brain-stem areas might be critical for the performance of conditioned reflexes. In 6 cats, large bilateral electrolytic lesions were placed in the medial diencephalon or mesencephalon and subsequently defined by histology. A tone served as conditional stimulus and foot-shock as unconditional stimulus. The EEG, respiration, and the EMG of the conditioned limb were recorded. Persistent low voltage, fast activity consistently reappeared in the EEG in 7 to 10 days and was seen even after destruction of over 100 mm 3 of the mesencephalon, including the reticular formation. Conditioned reflexes usually appeared only when such faster EEG patterns were present. Conditioned reflexes and discrimination between tones could occur, however, during relatively high voltage, slow activity in the cortical areas sampled. Extensive lesions essentially transecting the ascending reticular system or destroying the posterior hypothalamus did not preclude conditioning. In contrast, one animal could not be retrained when lesions were in the mamillary area. It is concluded that the medial mesencephalic systems are not essential for this type of conditioning and that, if any essential system does exist, it is located in the region of the mamillary bodies, field H 1 of Forel, center median, and the habenulopeduncular tract.