Previous studies have produced mixed results about the role of the ventral tegmental area, periaqueductal gray and parabrachial nucleus in morphine discriminations, perhaps owing to the considerably different methodologies used. The purpose of the present study was to compare the roles of these three brain areas using the same food-reinforced discrimination protocol, to determine whether the schedule of reinforcement influenced maximal substitution produced by site-specific morphine administration and to determine whether the time course of substitution differed by site of morphine administration. Rats were trained to discriminate 3.0 mg/kg subcutaneous morphine from saline under variable interval 15-s or fixed ratio 10 schedules of food reinforcement. Rats were then implanted with one cannula aimed at the lateral ventricle (intracerebroventricular) and one aimed at the ventral tegmental area, periaqueductal gray or parabrachial nucleus. Morphine discrimination curves were obtained by subcutaneous, intracerebroventricular and intrasite routes. When administered subcutaneously, morphine was equipotent in variable interval-trained and fixed ratio-trained rats, although it was more potent in fixed ratio-trained females than fixed ratio-trained males. When administered intracerebroventricularly, morphine (0.3–10 μg) engendered a maximum average of 63% drug-appropriate responding in both variable interval-trained and fixed ratio-trained rats; females showed significantly greater drug-appropriate responding than males, again under the fixed ratio but not under the variable interval schedule. In variable interval-trained rats, intrasite infusions of morphine (0.3–10 μg) produced maximal drug-appropriate responding of approximately 57% (ventral tegmental area), 56% (periaqueductal gray) and 41% (parabrachial nucleus); mean maximal substitution was slightly (≤17%) greater in fixed ratio-trained rats. When injected into the ventral tegmental area or periaqueductal gray, but not the parabrachial nucleus, naloxone methiodide (2 μg) significantly decreased drug-appropriate responding following 3.0 mg/kg subcutaneous morphine, in both variable interval-trained and fixed ratio-trained rats. The time course of the discriminative stimulus effects of morphine differed among the three brain sites: intraventral tegmental area morphine produced peak drug-appropriate responding by 15 min after injection, whereas the discriminative stimulus effects of intraperiaqueductal gray and intraparabrachial nucleus morphine peaked at approximately 60 min after injection. Taken together, these results indicate that ventral tegmental area, periaqueductal gray and parabrachial nucleus each play a role in the ability of morphine to function as a discriminative stimulus, regardless of the sex of the subject or the schedule under which the subjects are responding. Ventral tegmental area and periaqueductal gray, however, appear to be more critical than parabrachial nucleus in mediating the discriminative effects of systemic morphine in rats responding under a food reinforcement procedure. The pretreatment time and, to a lesser extent, the schedule of reinforcement are additional variables that should be considered when comparing the relative roles of different brain areas in drug discrimination.