BACKGROUND Nicotine and ethanol are frequently co-abused in men and women, but few studies compare common stimulus effects produced by these substances between males and females. OBJECTIVE This study compared the anxiety-like behavior induced by nicotine prior to and during ethanol withdrawal in intact male, sham-operated female, and ovariectomized (OVX) rats. METHODS Using an animal model of anxiety, the pentylenetetrazol (PTZ) drug-discrimination assay, rats were trained to discriminate PTZ (16 mg/kg, i.p.) from saline and were subjected to the following tests: (1) PTZ-lever selection at 12 h after termination of ethanol diet (4.5% for 10 days); (2) dose-response tests for nicotine (0.08-1.3 mg/kg) prior to ethanol and 1.5, 6, and 7 days after ethanol withdrawal. RESULTS (1) During acute ethanol withdrawal (12 h), more male rats (43.4%) responded on the PTZ lever than OVX (29%) or sham female (15.3%) rats. (2) For nicotine dose-response tests, more male rats (70%) selected the PTZ lever than OVX (37.5%) or sham female (50%) rats prior to ethanol. At 1.5 days, nicotine fully generalized to the PTZ stimulus in male (100%) and OVX (90%), but only partially in sham female (50%) rats. At 6 days and 7 days after ethanol withdrawal, the PTZ-lever selection decreased, but more male rats (78%) tended to respond on a PTZ lever than OVX (63.6%) or sham female rats (62.5%). CONCLUSIONS Acute nicotine produces anxiety-like behavior similar to that of PTZ in male and female rats, and this effect of nicotine is intensified during ethanol withdrawal in male and OVX rats, but not in sham female rats.