The cyclin-dependent kinase Cdk5 and DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of Mr 32 kDa)-dependent signaling have been implicated in the regulation of dopaminergic neurotransmission after chronic cocaine treatment. In this study, we examined if Cdk5 signaling participates in the behavioral and biochemical effect of acute and chronic methamphetamine (METH) treatment. We found that Cdk5 activity and the membrane fraction of p35 protein, a Cdk5 activator, in the ventral striatum increased transiently after an injection of 4 mg/kg METH, while intra-accumbens treatment with a Cdk5 inhibitor, roscovitine, prevented the acute METH-induced locomotor activation. The phosphorylation of DARPP-32 at both Thr75 and Thr34 was differentially regulated after acute METH treatment, but the levels of total Cdk5, p35, and DARPP-32 remained the same. To determine if Cdk5 signaling was associated with behavior sensitization to METH, rats that received repetitive injections of METH (4 mg/kg) for 14 consecutive days were analyzed at withdrawal day 7. The results indicate that Cdk5 activity and p35 translocation in the ventral striatum were upregulated in METH-sensitized rats; treatment with roscovitine in the nucleus accumbens effectively suppressed the 1 mg/kg METH-induced behavioral sensitization. Concomitantly, a decrease in the amount of PP-2A and DARPP-32 phosphorylation at Thr34, but an increase in phosphorylation of DARPP-32/Thr75, was observed in the ventral striatum of sensitized rats. The overall results demonstrate that Cdk5/p35 and downstream signaling in the ventral striatum play a critical role in the effects of acute METH treatment as well as the development of behavioral METH sensitization.