Estrogens regulate dendritic spine density, but the mechanism and significance of this effect for brain networks remain unknown. We used repetitive imaging over several days to investigate how 17β-estradiol affected the turnover and long-term behavior of dendritic spines in CA1 cells of hippocampal slice cultures. We find that 17β-estradiol and serum in the culture medium tightly regulated spine density by promoting an increase in the rate of new spine formation and their transformation into synapses, without affecting spine elimination or stability. New spines formed during a transient 17β-estradiol application were preferentially eliminated upon removal of the hormone, in contrast with pre-existing spines that remained unaffected. Our results reveal that 17β-estradiol transiently regulates the complexity of hippocampal circuits without causing major alterations of pre-existing networks. © 2010 Wiley Periodicals, Inc.