Psychostimulant effects are enhanced by ovarian hormones in women and female rodents. Estradiol increases behavioral responses to psychostimulants in women and female rats, although the underlying mechanism is unknown. This study utilized mice to investigate the time frame and receptor mediation of estradiol's enhancement of cocaine-induced behavior as mice enable parallel use of genetic, surgical and pharmacological methods. The spontaneous behavior of Sham and Ovariectomized (Ovx) female wildtype (WT) mice was determined during habituation to a novel environment and after cocaine administration. Ovx mice were replaced with vehicle (sesame oil) or 17β-estradiol (E2) for 2 days or 30 min prior to a cocaine challenge to investigate the time course of E2's effects. To examine receptor mediation of estradiol effects, Ovx mice replaced for 2 days with either the ERα-selective agonist PPT or the ERβ-selective agonist DPN were compared to Sham mice, and mice lacking either ERα (αERKO) or ERβ (βERKO) were compared to WT littermates. Ovx mice exhibited fewer ambulations during habituation than Sham females. Cocaine-induced increases in behavioral ratings were greater in Sham than in Ovx mice. Two days but not 30 min of E2 replacement in Ovx mice increased cocaine responses to Sham levels. PPT replacement also increased the cocaine response relative to vehicle- or DPN- treated Ovx mice. αERKO mice displayed modestly attenuated behavioral responses to novelty and cocaine compared to αWT littermates, but no behavioral differences were found between βERKO and βWT mice. These results suggest that E2 enhances cocaine-stimulated locomotion in mice predominantly through ERα.