Estrogens differentially modulate behavior in the adult female rodent. Voluntary exercise can also impact behavior, often reversing age associated decrements in memory processes. Our research group has published a series of papers reporting a deficit in the acquisition of an operant working memory task, delayed spatial alternation (DSA), following 17β-estradiol treatment to middle-aged ovariectomized (OVX) rats. The current study examined if voluntary exercise could attenuate the 17β-estradiol induced deficits on DSA performance. OVX 12-month old Long–Evans rats were implanted with a Silastic capsule containing 17β-estradiol (10% in cholesterol: low physiological range) or with a blank capsule. A subset of the 17β-estradiol and OVX untreated rats were given free access to a running wheel in their home cage. All rats were tested for 40 sessions on the DSA task. Surprisingly, we found running wheel access to impair initial acquisition of the DSA task in 17β-estradiol treated rats, an effect not seen in OVX untreated rats given running wheel access. This deficit was driven by an increase in perseverative responding on a lever no longer associated with reinforcement. We also report for the first time a 17β-estradiol induced impairment on the DSA task following a long intertrial delay (18-sec), an effect revealed following more extended testing than in our previous studies (15 additional sessions). Overall, running wheel access increased initial error rate on the DSA task in 17β-estradiol treated middle-aged OVX rats, and failed to prevent the 17β-estradiol induced deficits in performance of the operant DSA task in later testing sessions.