Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer. Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown. In this study we demonstrate that tamoxifen interacts directly with P-gp. Plasma membranes from P-gp-expressing cells bound [3H]tamoxifen in a specific and saturable fashion. A 180 kDa membrane protein in these membranes, labelled by the affinity analogue tamoxifen aziridine and azidopine, was shown to be P-gp. Tamoxifen reduced the binding of vinblastine and azidopine to P-gp, and tamoxifen increased [3H]vinblastine accumulation in P-gp-expressing cells to levels approaching those in non-P-gp-expressing cells. However, the cellular accumulation of [3H]tamoxifen itself was not influenced by the presence of P-gp. Thus, tamoxifen appears to reverse multidrug resistance by binding to P-gp and inhibiting the transport of cytotoxic drugs, but does not itself appear to be transported by the protein.