The importance of the dopaminergic system for proper brain activity is demonstrated by findings that alterations in this system lead to severe disabilities, including motor impairment observed in various neurological and psychiatric disorders. Although the roles of specific dopamine receptors in behaviour have been extensively investigated using pharmacological agents and knockout mice, non-specificity of ligands and compensatory molecular adaptations in mutated animals restrict the interpretation of the results. To overcome these limitations and further explore the role of the dopamine D2 and D3 receptors (D2R and D3R) in rats, we used lentivirus-mediated gene knockdown and overexpression to specifically manipulate expression levels of these genes in the rat nucleus accumbens (NAcc), a brain area important for spontaneous and induced locomotor responses. Lentiviruses, inducing expression of rat D2R or D3R, or efficient knockdown of either receptor by small hairpin (sh)RNAs, were stereotaxically injected into the NAcc. While knockdown of either receptor significantly reduced spontaneous locomotor activity in a novel but not in a habituated environment, D2R and D3R appeared to contribute in opposite ways to light-induced locomotor activity. D2R knockdown increased while D3R knockdown decreased locomotor activity in this test. Altogether, our findings suggest that D2R and D3R, expressed in the NAcc, have both shared and non-overlapping roles in transduction of alerting signals elicited by potentially important sensory and environmental cues.