Tamoxifen is widely used as an adjunct therapy for breast cancer. We hypothesized that hypoxia develops in tumors as a result of tamoxifen treatment because tamoxifen has been reported to be antiangiogenic and thrombogenic. MCF-7 breast tumors were grown under estrogenic stimulation in 4-6-week-old CD-1 nu/nu female mice. When the tumors were approximately 5 mm in diameter, 17beta-estradiol pellets were replaced with either placebo or tamoxifen-containing pellets. Two days later, tissue oxygenation was measured using immunohistochemical detection of binding of the 2-nitroimidazole EF5. Intravascular oxygen partial pressures were measured noninvasively by oxygen-dependent quenching of phosphorescence of an injected dye that is excited by light pulses. Tamoxifen treatment increased hypoxia in the tumors, as measured by EF5 binding (P = 0.01 by Mann-Whitney test). This observation was not dependent on the presence of tamoxifen-induced necrosis. Intravascular oxygen partial pressures were lower in tumors relative to surrounding normal tissue in tamoxifen-treated tumors as compared to placebo-treated tumors. In vitro, tamoxifen did not modify the oxygen-dependent metabolism of EF5, indicating that the increased EF5 binding in tamoxifen-treated tumors reflects a physiological decrease in tissue oxygenation. The clinical significance of these observations is discussed in the context of the sequencing of tamoxifen with other therapies, and in light of recent data suggesting that hypoxia may be associated with genetic changes resulting in a more aggressive tumor phenotype.