It has been proposed that nicotine-stimulated locomotor activity (LMA) and nicotine-induced dopamine (DA) release in the mesocorticolimbic DA system is partly regulated by glutamate receptors, particularly N-methyl-D-aspartate (NMDA) receptors. The functional characteristics of NMDA receptors depend on their subunit composition (NR1 in combination with NR2A-D). In the present study, we investigated the effect of the NR2B-selective NMDA receptor antagonist Ro 25-6981 [(+/-)-(R*,S*)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propanol] on nicotine-stimulated LMA and nicotine-induced DA release in the nucleus accumbens (NAcc) in rats. Ro 25-6981 (3 and 10 mg/kg i.p.) given 10 min prior to a high dose (0.6 mg/kg s.c.) or a subthreshold dose (0.1 mg/kg s.c.) of nicotine potentiated nicotine-stimulated LMA with no effect when administered alone. Similarly, administration of a low dose (0.05 mg/kg i.p.) of the noncompetitive NMDA receptor antagonist MK-801 (dizocilpine maleate) had no effect on LMA by itself but potentiated nicotine-induced (0.1 mg/kg) LMA. However, pretreatment with the competitive NMDA receptor antagonist CGP39551 [(E)-(+/-)-2-amino-4-methyl-5-phosphono-3-pentenoic acid ethyl ester] (10 mg/kg i.p.) did not potentiate the LMA effect of 0.1 mg/kg nicotine as seen with Ro 25-6981. In vivo microdialysis revealed a significant increase of DA release in the NAcc in response to nicotine (0.1 mg/kg s.c.). In analogy to our LMA data, Ro 25-6981 (10 mg/kg i.p.) significantly potentiated the nicotine-induced DA release, although it had no effect on DA release when given alone. The data suggest that, compared with other subunits of the NMDA receptor, the NR2B subunit might play a different role in the reinforcing effects of nicotine.