The dopamine D3 receptor is believed to play an important role in regulation of rodent locomotor behavior, and has been proposed as a therapeutic target for substance abuse, psychotic disorders, and Parkinson's disease. One model of dopamine D3 receptor function, based on studies utilizing D3 receptor knockout mice and D3 receptor-preferring agonists, proposes that D3 receptor stimulation is inhibitory to psychostimulant-induced locomotion, in opposition to the effects of concurrent dopamine D1 and D2 receptor stimulation. Recent progress in medicinal chemistry has led to the development of highly-selective dopamine D3 receptor antagonists. In order to extend our understanding of D3 dopamine receptor's behavioral functions, we determined the effects of the highly-selective dopamine D3 receptor antagonist NGB 2904 on amphetamine-stimulated and spontaneous locomotion in wild-type and dopamine D3 receptor knockout mice. NGB 2904 (26.0 μg/kg s.c.) enhanced amphetamine-stimulated locomotion in wild-type mice, but had no measurable effect in dopamine D3 receptor knockout mice. Of a range of doses (0.026 μg-1.0 mg/kg) given acutely or once daily for seven days, the highest dose of NGB 2904 (1.0 mg/kg) stimulated spontaneous locomotion in wild-type mice, but was without measurable effect in dopamine D3 receptor knockout mice. These behavioral effects of NGB 2904 contrast with those described for other highly D3 receptor-selective antagonists, which have not previously demonstrated an effect on spontaneous locomotor activity. In combination, these data add to the behavioral profile of this novel D3 receptor ligand and provide further support for a role for dopamine D3 receptor inhibitory function in the modulation of rodent locomotion. © 2007 Elsevier Inc. All rights reserved.