Ovarian folliculogenesis is characterized, in part, by the formation and expansion of the fluid-filled antrum. Development of this cavity requires water influx, which may occur by transcellular or pericellular transport mechanisms. To assess the contribution of these mechanisms to the water permeability of an antral follicle, the rate of ³H₂O and ¹⁴C-inulin (a complex sugar restricted to the extracellular compartment) uptake into isolated follicles was determined. The rate of H₂O movement was 3.5-fold greater than that of inulin, suggesting that water enters a follicle primarily by transcellular pathways. Preincubation of the follicles with 50 μm HgCl₂ [a nonspecific aquaporin (Aqp) inhibitor] decreased H₂O movement to levels seen with inulin, indicating that transcellular water movement is mediated through Aqp. To demonstrate the functional presence of Aqp in granulosa cells, we show that swelling in response to a hypotonic insult is attenuated by preincubation with 50 μm HgCl₂. Flow cytometry demonstrated the presence of Aqps−7, −8, and −9, thus identifying candidate Aqp potentially mediating water movement into antral follicles. These results suggest that water permeability of antral follicles occurs primarily through transcellular mechanisms, which may be mediated by Aqps −7, −8, and/or −9 in granulosa cells.