The dopamine D(3) receptor (D(3)R) is an important pharmacotherapeutic target for its potential role in psychiatric disorders and drug dependence. To further explore its function in rats, a regulatable lentivirus, Lenti-D3, holding the rat D(3)R cDNA, has been constructed as well as three nonregulatable lentiviruses, Lenti-D3-siRNA1, Lenti-D3-siRNA2 and Lenti-D3-siRNA3, expressing small hairpin RNAs, aimed at silencing D(3)R expression and specifically targeted against different regions of the D(3)R mRNA. In vitro, Lenti-D3 expressed D(3)R and could efficiently be blocked with Lenti-D3-Sils. These viruses were stereotaxically injected into the shell part of the nucleus accumbens (NAcc) and effects of passive cocaine delivery on locomotor activity were assessed. Manipulations of D(3)R levels induced changes in the locomotor stimulant effects of cocaine as compared to control treatment. Suppression of dopamine (DA) D(3)R in the NAcc by means of local knockdown (with Lenti-D3-Sils) increased locomotor stimulant effects, whereas its overexpression with Lenti-D3 drastically reduced them. The latter effects could be reversed when animals were fed doxycycline, which prevented lentiviral-mediated DA D(3)R overexpression in the NAcc. Gene expression assessed by quantitative RT-PCR confirmed very efficient gene knockdown in vivo in animals treated with Lenti-D3-Sils (> 93% silencing of D(3)R gene). Thus D(3)R expression significantly contributes to behavioural changes associated with chronic cocaine delivery.