Women are 60% more likely to suffer from an anxiety disorder than men. One hypothesis for this difference may be that females exhibit increased rates of fear generalization. Females generalize fear to a neutral context faster than males, a process driven, in part, by estrogens. In the current study, ovariectomized adult female Long-Evans rats were given acute injections of estradiol benzoate (15μg/0.1mL sesame oil) or sesame oil during a passive avoidance procedure to determine if estrogens increase fear generalization through an effect on fear memory acquisition/consolidation or through fear memory retrieval. Animals injected 1h prior to training generalized to the neutral context 24h later but not 7days after training. Generalization was also seen when injections occurred 24h before testing, but not when tested at immediate (1h) or intermediate (6h) time points. In Experiment 3, animals were injected with estrogen receptor (ER) agonists, PPT or DPN, to determine which ER subtype(s) increased fear generalization. Only the ERβ agonist, DPN, increased fear generalization when testing occurred 24h after injection. Our results indicate that estradiol increases fear generalization through an effect on fear memory retrieval mechanisms by activation of ERβ.