Background Attention-deficit/hyperactivity disorder (ADHD) may result from delayed establishment of corticolimbic circuitry or perturbed dopamine (DA) neurotransmission. Despite the widespread use of stimulants to treat ADHD, little is known regarding their long-term effects on neurotransmitter levels and metabolism. Cyclin-dependent kinase 5 (Cdk5) regulates DA signaling through control of synthesis, postsynaptic responses, and vesicle release. Mice lacking the Cdk5-activating cofactor p35 are deficient in cortical lamination, suggesting altered motor/reward circuitry. Methods We employed mice lacking p35 to study the effect of altered circuitry in vivo. Positron emission tomography measured glucose metabolism in the cerebral cortex using 2-deoxy-2-[ 18F] fluoro-d-glucose as the radiotracer. Retrograde dye tracing and tyrosine hydroxylase immunostains assessed the effect of p35 knockout on the medial prefrontal cortex (PFC), especially in relation to mesolimbic circuit formation. We defined the influence of Cdk5/p35 activity on catecholaminergic neurotransmission and motor activity via examination of locomotor responses to psychostimulants, monoamine neurotransmitter levels, and DA signal transduction. Results Here, we report that mice deficient in p35 display increased glucose uptake in the cerebral cortex, basal hyperactivity, and paradoxical decreased locomotion in response to chronic injection of cocaine or methylphenidate. Knockout mice also exhibited an increased susceptibility to changes in PFC neurotransmitter content after chronic methylphenidate exposure and altered basal DAergic activity in acute striatal and PFC slices. Conclusions Our findings suggest that dysregulation of Cdk5/p35 activity during development may contribute to ADHD pathology, as indicated by the behavioral phenotype, improperly established mesolimbic circuitry, and aberrations in striatal and PFC catecholaminergic signaling in p35 knockout mice. © 2010 Society of Biological Psychiatry.