The ability of addictive drugs to induce adaptations in mesolimbic dopamine (DA) activity offers an attractive neurobiological explanation for enhanced incentive motivation toward drug-associated stimuli in addiction. However, direct evidence supporting this is sparse. By tracking neurochemical activity within the mouse nucleus accumbens via microdialysis during repeated pairing of morphine with environmental stimuli, we reveal a predictive relationship between enhanced DA responses to morphine and subsequent preference towards a morphine-paired stimulus. A similar relationship for serotonin (5-HT) was observed, suggesting that these neuromodulatory systems work in concert. During expression of preference towards a morphine-paired stimulus, extracellular DA was not enhanced but was negatively associated with this behavior on a subject-by-subject basis. In contrast, avoidance of an aversively-paired stimulus (the opiate antagonist naloxone) was associated with enhanced extracellular DA levels, and also the balance between DA and 5-HT responses. These findings reveal a tangible predictive relationship between drug-induced neural adaptations and conditioned behavior, and emphasize that DA activity is not generalized to all subcomponents of behavior conditioned by addictive drugs. They further provide evidence for an active role of DA–5-HT interactions in the expression of learned behavior.