Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I_A, and a sustained current, I_K. Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I_A or I_K in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I_A and I_K were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I_A in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I_A regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I_A activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.