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Rationale: Male rats trained to associate a neutral odor or rodent jacket on a female with their post-ejaculatory reward state display a preference to ejaculate with females bearing the odor or jacket. This conditioned ejaculatory preference (CEP) can be shifted by systemic administration of the opioid antagonist naloxone (NAL) during training, such that NAL-trained males distribute their ejaculations to females without the cue, relative to saline (SAL)-trained males., Objective: The present study examined two brain sites, the medial preoptic area (mPOA) or ventral tegmental area (VTA), where the opioid reward state might be induced., Methods: Sexually naïve Long-Evans males were implanted with bilateral guide cannula aimed at either site before they underwent multi-ejaculatory conditioning trials at 4-day intervals with sexually receptive females that bore either an almond odor or rodent tethering jacket. Infusions of NAL (1 μl/side) or SAL (1 μl/side) were made prior to each conditioning trial. All males were infused with SAL prior to a final open-field choice test with two sexually receptive females, one scented and the other unscented, or one jacketed and the other unjacketed., Results: Males previously conditioned with SAL in either region showed significant CEP. In contrast, prior infusions of NAL to the mPOA shifted the preference towards the unfamiliar female, whereas prior infusions to the VTA abolished CEP for the odor. Subsequent detection of Fos protein induced by the cue showed that, relative to SAL-treated males, prior experience with NAL in the mPOA suppressed Fos in both the mPOA and VTA, whereas prior experience with NAL in to the VTA suppressed Fos in the VTA alone., Conclusions: Opioid antagonism in the mPOA produces a state of non-reward whereas in the VTA, it produces a state in which the odor does not acquire incentive properties.