Abstract An elevated plus-maze, used to identify anxiolytic effects of drugs (Pellow et al., 1985), reflected as increased open-arm exploration, was employed in cycling and ovariectomised rats, to determine the effect of diazepam on the cycle and, after ovariectomy, to investigate possible influences of treatment with oestradiol (10 or 100 μg/kg, s.c.) (acute: 1 day or subchronic: 3 days) to ovariectomised rats on the effect of diazepam (1 mg/kg, i.p.), studied 3, 24 and 72 hr after cessation of treatment. Cycling females, exhibiting 3 consecutive 4-day oestrous cycles and ovariectomised rats, 15 days after surgery, were used. There was a tendency for increased open-arm exploration by rats in proestrous and diestrous, as compared to the other phases of the cycle and to ovariectomised rats. Rats treated 24 hr after subchronic treatment with oestradiol also exhibited increased open-arm exploration. The anti-anxiety effect of diazepam was clearly shown in oestrous and metestrous but not in proestrous, diestrous or in ovariectomised rats. The effect of diazepam was abolished in rats tested 3 hr after acute small doses of estradiol and attenuated in rats tested 24 hr after subchronic administration of the hormone. The results suggest that oestradiol, administered in physiological doses, may have an inhibitory effect on the diazepam-induced anxiolysis.