Intravenous naloxone enhanced, and D-Ala2-Met-enkephalinamide (DALA) impaired, Pavlovian conditioned heart rate discrimination in rabbits (Oryctolagus cuniculus) during initial training trials, compared with saline; naloxone also delayed subsequent extinction of the discrimination. These effects of the opioid treatments on discrimination were abolished by parasagittal knife-cut lesions in the sublenticular substantia innominata that did not, themselves, impair discrimination. Both naloxone and DALA decreased the magnitude and altered the topography of bradycardiac conditioned responses, and the lesions also abolished these effects of the opioid treatments, but they did not alter the effect of naloxone to decrease bradycardiac orienting response magnitude. These findings suggest that fibers in the sublenticular area mediate specifically the effects of systemic opioids on associative functions during Pavlovian cardiac conditioning and extinction.