Recent studies have reported that rats raised in an enriched condition (EC) have decreased dopamine transporter (DAT) function and expression in medial prefrontal cortex (mPFC), as well as increased d-amphetamine-induced glutamate release in nucleus accumbens compared to rats raised in an isolated condition (IC). In these previous studies, DAT function and expression were evaluated using mPFC pooled from four rats for each condition to obtain kinetic parameters due to sparse DAT expression in mPFC. In contrast, accumbal glutamate release was determined using individual rats. The current study extends the previous work and reports on the optimization of DAT and serotonin transporter (SERT) functional assays, as well as cell surface expression assays using both mPFC and orbitofrontal cortex (OFC) from individual EC or IC rats. In addition, the effect of d-amphetamine on glutamate release in mPFC and OFC of EC and IC rats was determined using in vivo microdialysis. Results show that environmental enrichment decreased maximal transport velocity (Vmax) for [(3)H]dopamine uptake in mPFC, but increased Vmax for [(3)H]dopamine uptake in OFC. Corresponding changes in DAT cell surface expression were not found. In contrast, Vmax for [(3)H]serotonin uptake and cellular localization of SERT in mPFC and OFC were not different between EC and IC rats. Further, acute d-amphetamine (2mg/kg, s.c.) increased extracellular glutamate concentrations in mPFC of EC rats only and in OFC of IC rats only. Overall, these results suggest that enrichment produces long-lasting alterations in mPFC and OFC DAT function via a trafficking-independent mechanism, as well as differential glutamate release in mPFC and OFC. Rearing-induced modulation of DAT function and glutamate release in prefrontal cortical subregions may contribute to the known protective effects of enrichment on drug abuse vulnerability.