Chronic nicotine administration leads to neuroadaptations, an important process in nicotine and tobacco dependence for which treatments are limited. The cysteine pro-drug, N-acetyl-cysteine (NAC), is a promising glutamatergic agent that has shown some clinical efficacy in reducing nicotine use in humans. The purpose of this study was to examine NAC in two rodent models of nicotine dependence. NAC (0, 5, 20, 50 and 100 mg/kg) was examined on locomotor activity in groups of rats previously exposed to nicotine or saline. In the second experiment, NAC (0, 50 and 100 mg/kg i.p.) was evaluated against the discriminative stimulus effects of nicotine (0.2 mg/kg) using a two-lever procedure under a tandem schedule (VI10"-FR10) of food reinforcement. Pre-treatment with NAC in doses greater than 20 mg/kg attenuated the expression of conditioned hyperactivity when rats were placed in locomotor boxes previously paired with chronic nicotine administration. The same doses of NAC had modest effects in attenuating nicotine-stimulated hyperactivity in nicotine-treated or saline-treated rats tested in the same locomotor boxes. In the discrimination task, NAC did not generalise to the nicotine stimulus and nor did it modify the dose-response curve to nicotine, suggesting that NAC may not modify the subjective effects of nicotine. These results suggest NAC selectively attenuates conditioned responses to nicotine-paired stimuli without modifying nicotine-induced hyperactivity or the discriminative stimulus effects of nicotine. Thus, the study proposes that if NAC was to act in a similar selective manner in humans, the specific action of NAC to attenuate conditioned responses may limit its potential as a treatment to manage nicotine dependence.