AbstractDespite the prevalent expression of freezing behavior following Pavlovian fear conditioning, a growing body of literature suggests potential sex differences in defensive responses. Our study investigated how female defensive behaviors are expressed in different threat situations and modulated by the estrous cycle. We aimed to compare freezing and flight-like responses during the acquisition and retrieval of fear conditioning using two distinct unconditioned stimuli (US) in two different spatial configurations: (1) electrical footshock (FUS) in a small, conventional enclosure with a grid floor, and (2) a predator-like robot (PUS) in a spacious, open arena. Fear conditioning with FUS showed no substantial differences between male and female rats of two different estrous cycles (proestrus and diestrus) in the levels of freezing and flight. However, when PUS was employed, proestrus female rats showed significantly more flight responses to the CS during both acquisition and the retrieval compared to the male and diestrus female rats. Taken together, our findings suggest that hormonal influences on the choice of defensive strategies in threat situations are significantly modulated by both the type of US and the spatial configuration of the environment.