Only male zebra finches sing and several telencephalic song control regions exhibit sex differences in neuron number that presumably reflect effects of estrogen (E2) exerted during the first few posthatch weeks. That is, implanting females with E2 during this time masculinizes neuron number and instills the capacity for vocal behavior. In certain song regions, E2 masculinizes neuron number by preventing the naturally-occurring death of neurons, long after their production, migration and process outgrowth are complete. However, in the Higher Vocal Center (HVC), the cellular mechanisms by which E2 establishes sex differences in neuron number are poorly understood. In contrast with other song regions, HVC neurogenesis overlaps with sexual differentiation and the incorporation of new neurons is greater in young males and E2-treated females, than in normal females. However, it is not known whether E2 promotes the addition of HVC neurons by stimulating their production, specification, and/or survival. To address this issue we injected males and females with [3H]thymidine on days 15 and 16 to label a small group of sexually dimorphic HVC neuronal cohorts born during sexual differentiation. Afterwards, on day 17, females were implanted with Silastic pellets filled with estradiol benzoate (EB) or left empty. We report here that EB exposure on day 17 masculinized (increased) the number of neurons in the HVC at day 35 that were labeled by [3H]thymidine injections on days 15/16. Thus, EB was able to increase cell number among at least some HVC neuronal cohorts after their final division, implying estrogenic regulation of post-mitotic events.(ABSTRACT TRUNCATED AT 250 WORDS)