We examined chronic effects of 17β-estradiol (E₂β) on the responses of isolated rat anterior cerebral small arteries to vasoactive substances with special reference to endothelial function. Female Sprague-Dawley rats were separated into four groups: (1) sham-operated group (Sham), (2) sham-operated plus E₂β treated group (Sham+E), (3) ovariectomized group (OVX), (4) ovariectomized plus E₂β treated group (OVX+E). 5-Hydroxytryptamine (5-HT) (10⁻¹⁰–10⁻³ M) and U46619 (10⁻¹⁵–10⁻⁸ M) induced concentration-dependent contractions in the cerebral small arteries. The 5-HT- and U46619-induced contractions were not affected by pretreatment with 3×10⁻⁵ M N^ω-nitro-L-arginine methyl ester (L-NAME). No significant difference in high potassium (80 mM)- and the agonists-mediated contractions was observed among the four groups. Administration of acetylcholine (ACh) (10⁻⁹–10⁻³ M) and sodium nitroprusside (SNP) (10⁻⁸–10⁻³ M) caused dose-related relaxations in the cerebral small arteries precontracted by 10⁻⁸ M U46619. Chronic treatment with E₂β caused a significant potentiation of the ACh-induced relaxations in the Sham+E and OVX+E groups. The dose–response curve for ACh in the OVX group was quite similar to that obtained with the Sham group. The ACh-induced relaxation was reduced significantly by pretreatment with 3×10⁻⁵ M L-NAME, and an additional treatment with 10⁻³ M L-arginine reversed significantly the L-NAME–induced inhibition. The removal of endothelial cells produced a significant reduction of the ACh-induced relaxation. Indomethacin (10⁻⁵ M) did not alter the ACh-induced relaxation. The findings suggest that E₂β potentiates ACh-induced endothelium-dependent relaxation in rat anterior cerebral arteries and that the potentiation may be, in part, mediated by increasing production and release of endogenous NO from the endothelial cells.