P-glycoprotein, the multidrug resistance protein that actively transports a wide variety of lipophilic substrates out of cancer cells, has recently been described in some normal tissues, including the endothelium of the brain and testes. Here we show that P-glycoprotein is also expressed in ovarian endothelium. In ovarian capillaries, the immunolabeled protein was detected with two monoclonal antibodies to P-glycoprotein. It was shown to be membrane-bound and to transport a known P-glycoprotein substrate. Expression of P-glycoprotein in endothelial cells suggests that this transport protein plays a role in enhancing or restricting vascular permeability to lipophilic molecules. If it does, then its role may be predicted from its site of expression on the luminal or abluminal face of the capillary wall. In the region of the endothelial nucleus, endothelial membranes are sufficiently far apart that they can be distinguished at the light microscopic level. Confocal examination of tissue sections double labeled for P-glycoprotein and nuclei confirmed that, in brain, P-glycoprotein is expressed only on luminal membranes. This location is consistent with its putative role in protecting the neuropil from circulating lipophilic molecules. In both testicular and ovarian endothelium, however, P-glycoprotein is expressed on both luminal and abluminal membranes. This localization suggests that it acts to exclude P-glycoprotein substrates from the endothelial cells themselves.