During metestrus of the estrous cycle, a number of neutrophils infiltrate into the vaginal vault, presumably due to a neutrophil-specific chemokine, MIP-2, in mice. The physiological role of the infiltrating neutrophils, however, remains largely obscure. In this study we examined the effects of neutrophil depletion on the estrous cycle and steroid hormone levels. When mice were treated with an anti-Gr-1 mAb, they became neutropenic, as assessed as to the number of neutrophils in the peripheral blood. The estrous cycle of such mice was specifically blocked at diestrus irrespective of the phase at which the anti-Gr-1 mAb was administered. The blockade was reversible, because restoration of neutrophils to a normal level caused a restart of the cycle. Immunohistochemical analyses revealed that neutrophils were present mainly on the luminal surface and in the lumen at metestrus and to a lesser extent at diestrus but scarcely in the uterine cervix at any phase, and that the anti-Gr-1 mAb depleted neutrophils but not eosinophils in the vagina. The treatment with the anti-Gr-1 mAb significantly affected the serum 17beta-estradiol and progesterone levels at diestrus after the estrous cycle was blocked. Together, these results suggest that neutrophil infiltration into the vagina is critical in maintaining the estrous cycle through control of steroid hormone levels.