Activation of extracellular signal-regulated kinase (ERK) signaling is involved in drug-associated memory. We have previously demonstrated that activation of ERK in the nucleus accumbens via dopamine D1 and D2 receptors plays an important role in the expression of methamphetamine (METH)-associated conditioned place preference. However, it remains poorly understood how drug-associated cues induce relapse of drug-seeking behavior. Using a mouse model of cue-induced reinstatement of extinguished METH-seeking behavior, we investigated the role of ERK's activation in cue-induced relapsing behavior. Expression of the cue-induced reinstatement of METH-seeking behavior in mice was accompanied by the activation of ERK in the dorsomedial prefrontal cortex and hippocampus. Inhibition of ERK activation by systemic administration of SL327, a selective inhibitor of MAP kinase/ERK kinase (MEK), attenuated the expression of METH-associated cue-induced reinstatement, but had no effect on METH self-administration. Microinjection of PD98059, another specific MEK inhibitor, into the dorsomedial prefrontal cortex disrupted METH-associated cue-induced reinstatement, whereas intrahippocampal injection of the inhibitor had no effect. The MEK inhibitors had no effect on the cue-induced reinstatement of food-seeking behavior. Our findings suggest that the activation of ERK in the dorsomedial prefrontal cortex plays a determinant role in the cue-induced relapse of METH-seeking behavior.