The well known gender-related differences in drug action may partly be explained by changes in activity and expression of drug metabolising enzymes, but also by modulation of active drug transport systems (e.g. P-glycoprotein, Pgp) by sexual steroids, which is yet not well investigated. Because many women are using hormones (e.g. as oral contraceptives) we investigated the influence of different synthetic progestins on Pgp activity. Pgp inhibition of progesterone, medroxyprogesterone, chlormadinone, cyproterone, levonorgestrel, norethisterone, desogestrel, and norgestimate was measured in vitro in two Pgp over-expressing cell lines (L-MDR1, P388/dx cells) and the corresponding parental cell lines by means of calcein assay, and ex vivo in human peripheral blood mononuclear cells (PBMCs) by rhodamine123 efflux. For most progestins tested, concentrations needed to double baseline fluorescence (f2) in L-MDR1 cells were similar to that of the potent Pgp inhibitor quinidine, whereas levonorgestrel and norethisterone did not reach f2. The results in P388/dx cells essentially confirmed our findings in L-MDR1 cells. Additionally, Pgp inhibitory activity of all progestins tested was also shown ex vivo in PBMCs. The potent Pgp inhibition by several synthetic progestins in vitro and ex vivo suggests that such an interaction might be clinically relevant despite generally low plasma concentrations of progestins. The results may be of particular importance for Pgp substrates, such as protease inhibitors and chemotherapeutic agents, for which intracellular concentrations are critical.