Recent work in zebra finches suggests that genes and hormones may act together to masculinize the brain. This study tested the effects of exogenous estradiol (E2) on 17β-hydroxysteroid dehydrogenase type IV (HSD17B4) and the co-localization of HSD17B4 and androgen receptor (AR) mRNA. We asked three primary questions: First, how does post-hatching E2 treatment affect HSD17B4 mRNA expression in males and females? Second, is this gene expressed in the same cells as AR. Third, if so does E2 modulate co-expression? Female finches implanted with 50 μg of E2 on the third day post-hatching showed a significant increase in the density of cells expressing HSD17B4 and AR in HVC at day 25. Co-localization of AR cells that also expressed HSD17B4 was high across groups (>81%). We found significant sex differences in co-localization in both the HVC and Area X of control animals, with males showing a higher percentage of cells expressing AR mRNA that also expressed HSD17B4 in comparison to females. However, although E2 treatments significantly increased the number of cells expressing HSD17B4 mRNA and AR mRNA in the HVC of females, the percentage of HSD17B4 cells co-expressing AR was reduced in HVC and Area X in E2-treated animals. These results lend support to the hypothesis that genes and hormones may act in concert to modulate the sexually differentiation of the zebra finch song system. Further, the data suggest that a single hormonal mechanism cannot mimic the complex development of male singing behavior and associated song nuclei.