This study tested the effects of timing and duration of estradiol (E2) treatment, factors that are clinically relevant to hormone replacement in perimenopausal women, on social behavior and expression of genes in brain regions that regulate these behaviors. Female rats were ovariectomized (OVX) at 1 year of age, roughly equivalent to middle-age in women, and given E2 or vehicle for different durations (3 or 6 months) and timing (immediately or after a 3-month delay) relative to OVX. Social and ultrasonic vocalization (USV) behaviors were assessed at the 3 and 6 month timepoints, and the rats' brains were then used for gene expression profiling in hypothalamus (supraoptic nucleus, paraventricular nucleus), bed nucleus of the stria terminalis, medial amygdala, and prefrontal cortex using a 48-gene qPCR platform. At the 3-month post-OVX testing period, E2 treatment significantly decreased the number of frequency-modulated USVs emitted. No effects of hormone were found at the 6-month testing period. There were few effects of timing and duration of E2 in a test of social preference of a rat given a choice between her same-sex cagemate and a novel conspecific. For gene expression, effects of timing and duration of E2 were region-specific, with the majority of changes found for genes involved in regulating social behavior such as neuropeptides (Oxt, Oxtr & Avp), neurotransmitters (Drd1, Drd2, Htr2a, Grin2d & Gabbr1), and steroid hormone receptors (Esr2, Ar, Pgr). These data suggest that the mode of E2 treatment has specific effects on social behavior and expression of target genes involved in the regulation of these behaviors.