In general, the discriminative stimulus effects of drugs are examined following s.c. or i.m. administration; this may not be predictive of the effects of drugs that are abused by other means. Nicotine is primarily inhaled in the form of cigarette smoke. Here, two drug discrimination assays were used to compare the effects of nicotine administered i.v. or s.c. in rhesus monkeys responding under an FR5 schedule of stimulus shock termination. In monkeys discriminating 1.78 mg/kg nicotine s.c. (n=5), the ED50 of nicotine was 0.41 mg/kg. In monkeys discriminating 0.032 mg/kg nicotine i.v. (n=4), the ED50 of nicotine was 0.0067 mg/kg. In 3 of these monkeys, a dose of 3.2 mg/kg nicotine s.c. was required to substitute for the i.v. discriminative stimulus; up to 5.6 mg/kg nicotine did not substitute in the fourth monkey. The nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine antagonized the discriminative stimulus effects of nicotine in both discriminations; 1 mg/kg mecamylamine produced rightward shifts of the nicotine dose‐effect function of 3.2‐fold in the s.c. discrimination and a minimum 15‐fold in the i.v. discrimination. A dose of 3.2 mg/kg of the β2 subtype‐selective nAChR antagonist dihydro‐β‐erythroidine (DHβE) did not significantly antagonize the discriminative stimulus effects of nicotine in the s.c. discrimination. However, when combined with the training dose (0.032 mg/kg) of nicotine in the i.v. discrimination, DHβE dose‐dependently decreased discriminative stimulus effects (ED50=0.17 mg/kg). These results indicate there is a critical difference in the involvement of the β2 subtype of nAChR between the two discriminations that cannot be explained by the difference in training dose. Supported by USPHS Grant DA25267.