Methylphenidate (MP) is widely used to treat attention deficit hyperactivity disorder (ADHD). Variable number of tandem repeats polymorphisms in the dopamine D4 receptor (D(4)) gene have been implicated in vulnerability to ADHD and the response to MP. Here we examined the contribution of dopamine D4 receptors (D4Rs) to baseline brain glucose metabolism and to the regional metabolic responses to MP. We compared brain glucose metabolism (measured with micro-positron emission tomography and [(18)F]2-fluoro-2-deoxy-D-glucose) at baseline and after MP (10 mg/kg, i.p.) administration in mice with genetic deletion of the D(4). Images were analyzed using a novel automated image registration procedure. Baseline D(4)(-/-) mice had lower metabolism in the prefrontal cortex (PFC) and greater metabolism in the cerebellar vermis (CBV) than D(4)(+/+) and D(4)(+/-) mice; when given MP, D(4)(-/-) mice increased metabolism in the PFC and decreased it in the CBV, whereas in D(4)(+/+) and D(4)(+/-) mice, MP decreased metabolism in the PFC and increased it in the CBV. These findings provide evidence that D4Rs modulate not only the PFC, which may reflect the activation by dopamine of D4Rs located in this region, but also the CBV, which may reflect an indirect modulation as D4Rs are minimally expressed in this region. As individuals with ADHD show structural and/or functional abnormalities in these brain regions, the association of ADHD with D4Rs may reflect its modulation of these brain regions. The differential response to MP as a function of genotype could explain differences in brain functional responses to MP between patients with ADHD and healthy controls and between patients with ADHD with different D(4) polymorphisms.