ABSTRACT The endocannabinoid system has been implicated in the motivational effects of nicotine and nicotine-associated stimuli but the neural circuitry underlying tobacco addiction is not fully characterised. The present study aimed to establish a second-order schedule of nicotine reinforcement to compare the role of the endocannabinoid system in nicotine- and cue-maintained responding. The male rats were successfully trained to respond on a second-order schedule [FR5 (FR5: S) or FI 10′ (FR3: S)] under which presentation of the CS (brief light oscillation) was intermittently reinforced by nicotine (0.03 mg/kg/infusion). The relative contribution of nicotine and the CS towards responding was then compared. Nicotine and the CS were only able to independently maintain responding to similar level under the [FI 10′ (FR3: S)] schedule, which was subsequently employed to examine the effects of the selective CB1 receptor antagonist AM251. AM251 (0.1, 0.3 and 1 mg/kg, intraperitoneal [i.p.]) was used to examine the role of endocannabinoids in responding under the second-order schedule and responding maintained by independent presentation of nicotine and the CS. Responding under the second-order schedule was dose-dependently attenuated by AM251, whereas responding for independent presentation of nicotine and the CS was not affected. The establishment of second-order schedules of nicotine reinforcement in rodents highlighted the utility of such schedules for investigation of the neurobiology that underlies nicotine- and cue-maintained behaviour. Additionally, the role of CB1 receptors in nicotine-motivated behaviours was extended to those controlled under a second-order schedule.