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Recent research examining Pavlovian appetitive conditioning has extended the associative properties of nicotine from the unconditioned stimulus or reward to include the role of a conditional stimulus (CS), capable of acquiring the ability to evoke a conditioned response. To date, published research has used presession extravascular injections to examine nicotine as a contextual CS in that appetitive Pavlovian drug discrimination task. Two studies in the current research examined whether a nicotine CS can function discretely, multiple times within a session using passive iv infusions. In Experiment 1, rats readily acquired a discrimination in conditioned responding between nicotine and saline infusions when nicotine was selectively paired with sucrose presentations. In Experiment 2, rats were either trained with nicotine paired with sucrose or explicitly unpaired with sucrose. The results showed that rats trained with explicitly unpaired nicotine and sucrose did not increase dipper entries after the infusions. Nicotine was required to be reliably paired with sucrose for control of conditioned responding to develop. Implications of these findings are discussed in relation to tobacco addiction, learning theory, and pharmacology.