Many estrogen-replacement studies use ovariectomized animals as controls. However, ovariectomy greatly increases body weight and can enhance the peripheral synthesis of estrogen. Tamoxifen is commonly used as an antiestrogen, but it may elicit mixed agonist or antagonist actions. The aim of our study was to compare vascular function in mesenteric arteries among groups of rats with low estradiol levels. The groups (n = 5, each) of Sprague-Dawley rats were cycling (diestrus), ovariectomized (OVX), OVX + tamoxifen (OVX-T), OVX + 4-hydroxyandrostene-3,17-dione, an aromatase inhibitor (OVX-A) to prevent peripheral synthesis of estrogen, and control-fed OVX to prevent excess weight gain. Body weight was significantly elevated in only the non-control-fed OVX group. Estrogen levels were significantly greater in the cycling rats compared with the other groups, whereas uterine weights were significantly reduced in only the OVX-A and control-fed OVX groups. Methacholine relaxation was blunted only in the OVX-A and control-fed OVX groups, suggesting a possible estrogenic influence in the non-control-fed OVX and OVX-T groups. These data indicate the potential for confounding factors to decrease the efficacy of OVX controls.