This study investigated the effects of racemic methadone (MET) on P-glycoprotein (P-gp) activity in cell culture. MET showed no differential rates of passage between the basolateral to apical (B to A) and apical to basolateral (A to B) direction across Caco-2 cell monolayers in a transwell system. MET transport in either direction was not importantly influenced by the P-gp inhibitor verapamil. However, MET was a potent inhibitor (IC(50) = 7.5 microM) of rhodamine123 B to A transport across Caco-2 cell monolayers, causing a reduction to 25% of control at 100 microM MET. In this model of Caco-2 monolayers, rates of MET passage between B to A and A to B directions could not be distinguished. However, MET can inhibit P-gp activity at intraluminal concentrations that might be achieved clinically. This may lead to increased bioavailability of coadministered compounds.