Withdrawal from opioids can induce a state of anxiety and irritability. This negative state can facilitate continued drug taking, as the administration of opioids can alleviate unpleasant symptoms associated with acute and protracted withdrawal. It is, therefore, of interest to investigate factors that can contribute to the severity of anxiety during periods of abstinence. One such factor is the fluctuation of ovarian hormones. Evidence from a non-opioid drug indicates that estradiol increases, while progesterone decreases anxiety during withdrawal. However, no work has yet studied how ovarian hormones might influence the severity of anxiety during withdrawal from opioids. To explore this, we ovariectomized female rats and provided a four-day repeating cycle of ovarian hormone administration (Day 1: estradiol, Day 2: estradiol, Day 3: progesterone, Day 4: peanut oil). Male rats were given sham surgeries and administered peanut oil daily in lieu of hormone replacement. All rats received twice daily injections of morphine (or 0.9 % saline) for 10 days total at a dose that doubled every two days (2.5 mg/kg, 5 mg/kg, 10 m/kg, 20 mg/kg, 40 mg/kg). Rats underwent spontaneous withdrawal and were tested for anxiety-like behaviors 12 and 108 h after the last morphine treatment. At 12 h, morphine-withdrawn female rats treated with estradiol on the day of testing displayed significantly more anxiety-like behavior in light-dark box testing than female morphine-withdrawn and (marginally) male morphine-withdrawn rats receiving vehicle that day. Somatic withdrawal behaviors (wet dog shakes, head shakes, writhing) were also taken every 12 h through 108 h. We found no meaningful contribution of sex or hormone for these measures. This study is the first of its kind to provide evidence that ovarian hormones influence anxiety-like behavior during morphine withdrawal.Copyright © 2023. Published by Elsevier Inc.