BACKGROUND: Estetrol (E4) is a pregnancy-specific estrogenic steroid hormone produced by the human fetal liver in both male and female fetuses. During pregnancy, E4 plasma values increase exponentially until parturition and decrease thereafter. The synthesis of E4 in the liver of a newborn ceases during the first weeks after birth. MATERIALS AND METHODS: Here we report the effect of E4 on the initiation and growth of mammary tumors chemically induced by 7,12 dimethylbenz(a)anthracene (DMBA) in female Sprague-Dawley rats in two different protocols. Two prevention studies to test the effect on initiation and growth of induced tumors and one intervention study to test the effect on tumor growth were performed. In the prevention studies, the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg was investigated. In the intervention study, oral doses of 1, 3 and 10 mg/kg E4 were used. The anti-estrogen tamoxifen (TAM) and ethinylestradiol (EE) were used as reference compounds. In all studies, a group with ovariectomized animals (OVX) was included. RESULTS: In the prevention studies, 2.5 mg and 3 mg/kg E4 showed a significant effect on the number and growth of induced tumors by DMBA, and the effects were comparable to those of TAM, whereas EE had no effect. In the intervention study, the effect of a high dose of E4 (10 mg/kg) on tumor number was similar to that of OVX and better than TAM and high-dose EE. The 3 mg/kg E4 had an effect comparable to high-dose EE. The treatment effects were largely due to complete regression of existing tumors. CONCLUSIONS: The natural fetal estrogen E4 prevents tumor initiation by DMBA and inhibits the growth of existing DMBA-induced tumors.