Estrogens are known to increase running wheel activity of rodents primarily by acting on the medial preoptic area (mPOA). The mechanisms of this estrogenic regulation of running wheel activity are not completely understood. In particular, little is known about the separate roles of two types of estrogen receptors, ERα and ERβ, both of which are expressed in mPOA neurons. In the present study the effects of continuous estrogen treatment on running wheel activity were examined in male and female mice specifically lacking either the ERα (αERKO) or the ERβ (βERKO) gene. Mice were gonadectomized and 1 wk later implanted with either a low dose (16 ng/d) or a high dose (160 ng/d) of estradiol benzoate (EB) or with a placebo control pellet. Home cage running wheel activity was recorded for 9 d starting 10 d after EB implants. The same mice were also tested for open field activity before and after EB implants. In both female and male αERKO mice, running wheel activity was not different from that in corresponding wild-type (αWT) mice in placebo control groups. In both females and males it was increased by EB only in αWT, not αERKO, mice. In βERKO mice, on the other hand, both doses of EB equally increased running wheel activity in both sexes just as they did in βWT mice. Absolute numbers of daily revolutions of EB-treated groups, however, were significantly lower in βERKO females compared with βWT females. Before EB treatment, gonadectomized αERKO female were significantly less active than αWT mice in open field tests, whereas βERKO females tended to be more active than βWT mice. In male mice there were no effect of ERα or ERβ gene knockout on open field activity. Unlike its effect on running wheel activity, EB treatment induced only a small increase in open field activity in female, but not male, mice. These findings indicate that 1) in both sexes estrogenic regulation of running wheel activity is primarily mediated through the ERα, not the ERβ; and 2) hormone/genotype effects are specific to the type of locomotor activity (i.e. home cage running wheel activity and open field activity) measured.