Previous studies have shown that uptake of the lipophilic opioid, fentanyl, by pulmonary endothelial cells occurs by both passive diffusion and carrier-mediated processes. To evaluate if the latter mechanism also exists in brain endothelium, transport of [3H]fentanyl was examined in primary cultured bovine brain microvessel endothelial cell (BBMEC) monolayers. Uptake of fentanyl appears to occur via a carrier-mediated process as uptake of [3H]fentanyl by BBMECs was significantly inhibited in a dose-dependent manner by unlabeled fentanyl. Fentanyl uptake was also significantly inhibited by either 4 degrees C or sodium azide/2-deoxyglucose, suggesting that carrier-mediated uptake of fentanyl was an active process. Fentanyl was also tested to determine whether it might be a substrate of the endogenous blood-brain barrier efflux transport system, P-glycoprotein (P-gp). Release of [3H]fentanyl or rhodamine 123, a known substrate of P-gp, previously loaded in the BBMECs was studied in the presence or absence of either fentanyl or verapamil, a known competitive inhibitor of P-gp. Both fentanyl (10 microM) and verapamil (100 microM) decreased release of rhodamine 123 from BBMECs, indicating that fentanyl is a substrate of P-gp in the BBMECs. This was further supported by the observation that uptake of [3H]fentanyl was significantly increased in Mg2+-free medium, a condition known to reduce P-gp activity. However, release of [3H]fentanyl was significantly increased when incubated with either unlabeled fentanyl or verapamil. These results suggest that the active P-gp-mediated extrusion of fentanyl in these cells is overshadowed by an active inward transport process, mediated by an as yet unidentified transporter. In addition, verapamil was shown to be a substrate of both P-gp and the fentanyl uptake transporter.