Male Sprague-Dawley rats that were naive or that had been treated with five daily saline or cocaine injections (15 mg/kg i.p.) were subsequently challenged with an injection of cocaine, and extracellular dopamine content in the medial prefrontal cortex (mPFC) was measured using in vivo microdialysis. Cocaine challenge increased extracellular dopamine levels from base line in all three groups of rats, but the augmentation was significantly reduced in the cocaine-pretreated group, compared with the saline-pretreated group. In contrast, mPFC dopamine levels were not different among groups after challenge with systemic d-amphetamine. To test whether repeated cocaine treatment led to altered releasability of dopamine from mPFC terminals, challenge with KCI (10, 30 or 100 mM) or d-amphetamine (3, 30 or 300 microM) was made via infusion through the dialysis probe into the mPFC. No differences in dopamine levels were found between treatment groups for either drug at any dose. To determine whether the effects of cocaine were mediated by local actions within mPFC dopamine terminals, a cocaine challenge was administered through the microdialysis probe (1, 10 or 100 microM). In contrast to the systemic cocaine challenge, local infusion of cocaine elicited a significant increase in daily cocaine-pretreated rats, compared with saline-pretreated controls, at the lowest dose tested, with no differences at the higher two doses. In summary, daily cocaine-pretreated rats demonstrated a suppressed mPFC dopamine response to subsequent systemic, but not local, cocaine challenge. The results suggest that this apparent tolerance is not due to altered releasability of dopamine from mPFC terminals and may rely on altered afferent regulation of mesocortical dopamine neurons.