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The female reproductive cycle is orchestrated by cyclical and coordinated hormonal changes under the direction of the hypothalamic–pituitary–gonadal (HPG) axis. Any disruption of the HPG axis may lead to functional and structural alterations in the female reproductive system. Test article–related disturbances in the estrous cycle can be recognized in nonclinical toxicity studies by staging the cycle based on microscopic evaluation of female reproductive organs. In chronic rat toxicity studies, an additional complication is the development of reproductive senescence, which is associated with natural alterations in the reproductive cycle leading to changes in the female reproductive system that can potentially be confused with test article effects. The current article describes the features of persistent estrus, one stage of reproductive senescence, in middle-aged Sprague-Dawley rats and discusses elements to help differentiate senescence from induced effects.