Exposure of the fetus and placenta to maternal glucocorticoids is normally limited by the "placental glucocorticoid barrier", which consists primarily of placental 11-hydroxysteroid dehydrogenase type 2 (11-HSD2)-mediated conversion of cortisol to the biologically inactive cortisone. Studies in the rodent brain show that P-glycoprotein (P-gp) is also an important physiological regulator of glucocorticoid access to the glucocorticoid receptor (GR) in target cells, since it exports cortisol back into peripheral circulation against a concentration gradient. Whether P-gp of placental origin also has this capacity is unknown. Therefore, we used the human placental choriocarcinoma cell line BeWo and its daughter cell line, BeWoMDR, virally-transduced with P-gp, to assess whether placental P-gp regulates access of glucocorticoids to the GR. Quantitative PCR showed that BeWoMDR cells express 10-fold higher levels of P-gp mRNA than BeWo cells, and syncytialization increased P-gp mRNA by 7-fold. Elevated P-gp expression in BeWoMDR cells reduced activation of the GR by dexamethasone and cortisol (10^-9 M to 10^-6 M) to around 40% of that in BeWo cells. Inhibition of P-gp-mediated glucocorticoid efflux by cyclosporin A (CsA) in BeWoMDR cells returned GR activation to levels similar to those in BeWo cells. Diffusion of dexamethasone across BeWoMDR monolayers occurred at a slower rate than that across BeWo monolayers, but this difference was eliminated by CsA. These data support the hypothesis that P-glycoprotein contributes to the placental glucocorticoid barrier. Thus, 11-HSD2 and P-glycoprotein may act in unison to reduce fetal and placental exposure to maternal glucocorticoids and thereby minimize their growth inhibitory actions.