The aim of this study was to examine the state of anxiety and the 17beta-estradiol and progesterone levels in rats tested in the elevated plus-maze during the four phases of the estrous cycle. Male rats, female rats during each of the four phases of the estrous cycle, ovariectomized rats, and diestrus female rats treated with estradiol were tested in the elevated plus-maze between 8:00 and 10:00 a.m. Blood was collected from all rats for the determination of 17beta-estradiol and progesterone levels. Female rats in the proestrus group spent more time in the open arms than diestrus rats (P<.05). There were no significant differences in the percentage of entries into the open arms or in the number of entries into the closed arms among the phases of the estrous cycle or between males and normal or ovariectomized females. Serum estradiol levels were higher (P<.05) during proestrus compared to estrus, metestrus, and diestrus in control and plus-maze tested female rats, but there were no significant differences in progesterone levels. Treating diestrus female rats with estradiol to produce estradiol plasma concentrations similar to those seen during proestrus abolished the difference in the percentage of time spent in the open arms by proestrus and diestrus rats. Since the time spent in the open arms of the plus-maze is inversely related to anxiety, we conclude that the anxiety levels of female rats were lower in proestrus than during diestrus, and that the levels of estradiol modulate this response.