Adult female rats with high levels of circulating estradiol are biased to use a place strategy to solve an ambiguous spatial navigation task and those with low levels are biased to use a response strategy. We examined the development of this hormonal modulation of strategy use by training juvenile female rats on an ambiguous navigation task and probing them for strategy use at postnatal day (PD) 16, 21, or 26, after administration of 17 beta-estradiol or oil 48 and 24 h prior to testing. We found that rats could use either strategy successfully by PD21 but that estradiol did not bias rats to use a place strategy until PD26. In order to evaluate the stability of this effect over multiple navigation experiences, we retested oil-treated juveniles three times during adulthood. On the first adult navigation experience, rats were significantly more likely to use the same navigation strategy they used as juveniles, regardless of current estrous cycle phase. On the second and third adult tests, after rats had more experience with the task, previous navigation experience did not predict strategy use. Rats in proestrus were significantly more likely to use a place strategy while rats in estrus and diestrus did not appear to have a group bias to use either strategy. These results suggest that estradiol can modulate spatial navigation strategy use before puberty but that this effect interacts with previous navigation experience. This study sheds light on when and under what circumstances estradiol gains control over spatial navigation behavior in the female rat.