The ATP-sensitive potassium (K_ATP) channels couple intracellular metabolism to membrane potential. They are composed of Kir6.x and SUR subunits, and are expressed in hypothalamic neurons that project to GnRH neurons. However, their roles in regulating GnRH secretion have not been determined. The present study first tested whether K_ATP channels regulate pulsatile GnRH secretion, as indirectly reflected by pulsatile luteinizing hormone (LH) secretion. Ovariectomized rats received s.c. capsules containing either oil, estradiol-17 (E₂), progesterone (P), or E₂+P at 24h prior to blood sampling. Infusion of the K_ATP channel blocker tolbutamide into the 3^rd ventricle resulted in increased LH pulse frequency in animals treated with E₂+P but was without effect in all other groups. Coinfusion of tulbutamide and the K_ATP channel opener diazoxide blocked this effect, while diazoxide alone suppressed LH. Effects of steroids on Kir6.2 and SUR1 mRNA expression were then evaluated. After 24hr treatment, E₂+P produced a modest but significant increase in Kir6.2 expression in the POA, which was reversed by progesterone receptor antagonism with RU486. Neither SUR1 in the POA nor both subunits in the MBH were altered by any steroid treatment. After 8d treatment, Kir6.2 mRNA levels were again enhanced by E₂+P, but to a greater extent in the POA. Our findings demonstrate that 1) blockade of preoptic/hypothalamic K_ATP channels produces an acceleration of the GnRH pulse generator in a steroid-dependent manner, and 2) E₂+P stimulate Kir6.2 gene expression in the POA. These observations are consistent with the hypothesis that the negative feedback actions of ovarian steroids on the GnRH pulse generator are mediated, in part, by their ability to up-regulate K_ATP channel subunit expression in the POA.