Estrogen has effects on activity levels and emotional reactivity in both humans and rats. In a recent study conducted in ovariectomized (OVX) C57BL/6 (C57) mice we found that treatment with estradiol benzoate (EB) increased anxiety, fear learning, and running wheel activity relative to vehicle control (Veh). The present study was conducted to examine the stability of these findings across mouse strains (C57 and Swiss-Webster; SW), to get a better sense of the magnitude of the anxiety response by reducing baseline anxiety levels, and to discover if EB affects activity levels in a safe environment other than the home-cage running wheel. Mice of both strains treated with EB (s.c. implant, 25 microg in sesame oil, which enters the body over 5 weeks) were more anxious than Veh animals in the open field, elevated plus, and dark-light transition tests. SW animals were less anxious than C57 in the elevated plus. EB-treated animals of both strains were more active in the running wheel than Veh animals, and more active in the test of spontaneous activity in the home cage. EB-treatment also increased fear learning in a step-down avoidance task. EB appears to have a consistent but moderate effect in elevating anxiety and in increasing fear learning in two strains of mice. It is also involved in increasing activity in two different types of locomotion in the safer home cage. We conclude that these results of increased anxiety/fear and increased activity are suggestive of a general increase in arousal, with both sets of responses increasing the likelihood of reproductive behaviors occurring only when the environment predicts success.