Acute tolerance to effects of nicotine plays an important role in nicotine dependence, but the mechanism underlying these effects is unclear. Drug discrimination was used in the current study to examine the impact of nicotine pretreatment on sensitivity to the discriminative stimulus effects of nicotine and the FDA-approved smoking cessation pharmacotherapy varenicline. Rhesus monkeys (n = 4) discriminated 0.032 mg/kg nicotine base iv from saline under an FR5 schedule of stimulus-shock termination. Both nicotine and varenicline increased drug-appropriate responding; ED50 values (95% confidence limits) were 0.0087 [0.0025, 0.030] and 0.028 [0.0096, 0.082] mg/kg, respectively. Additional pretreatment injections of the training dose of nicotine (0.032 mg/kg, iv) produced tolerance to its discriminative stimulus effects and the magnitude of this effect was related to the number of pretreatment injections administered. Two pretreatment injections of the training dose of nicotine (0.032 mg/kg, iv) produced a 5.4-fold rightward shift in the nicotine dose-response function and a sevenfold rightward shift in the varenicline dose-response function. The duration of tolerance under these conditions was less than 60 min. These results demonstrate that tolerance to the discriminative stimulus effects of nicotine can be produced by acute nicotine exposure. Acute cross-tolerance from nicotine to varenicline is consistent with similar actions at nAChRs, and suggests that conditions resulting in acute nicotine tolerance could impact sensitivity to other nAChR agonists.