To examine the role of estradiol in hippocampal-dependent spatial memory in women, 86 female undergraduates were tested in a virtual Morris water task (VMWT), a virtual radial arm maze (VRAM), and a mental rotation task (MRT) within a single daily session. The VMWT and RAM were also administered 24 h later to examine the effects of estradiol on memory consolidation. Women on oral contraceptives (OCs) or those who were naturally cycling and exhibited low estradiol (LE) or high estradiol (HE), as determined by salivary assays, were included. At the start of day two, the HE group showed superior spatial reference memory on the VMWT relative to the LE group, as evidenced by significantly shorter distances navigating to the hidden platform. The LE group also had the poorest probe trial performance at the start of day two compared to both other groups. There were no group differences in performance on the RAM or MRT. These results provide support for estradiol’s role in the consolidation of spatial reference memory in women, and emphasize the differential sensitivities of various virtual memory tasks in assessing spatial memory function in women.