The objectives of this study were to determine (1) the effect of early nutrition on reproductive development and (2) response of reproductive tissues to estradiol (E₂) in pre-pubertal heifers. Holstein heifer calves (n = 23; 7 d of age) were fed either a restricted milk replacer diet (20% CP, 20% fat, 454 g/d) or accelerated diet (28% CP, 25% fat 1,333 g/d) for 8 wks. After the initial 8 wks, heifers were partitioned into a 2 × 2 factorial design and treated for an additional 2 wks accordingly: restricted diet + placebo implant (CON; n = 6), accelerated diet + placebo (ACC; n = 5), restricted diet + E₂ implant (CONEST; n = 6) and accelerated diet + E₂ (ACCEST; n = 6). Estrogen receptor α (ERα) and β (ERβ) gene expression was determined from RNA isolated from harvested endometrial, ovarian, and hypothalamic tissues using Real-Time qPCR. Fold changes were analyzed using the GLM procedure in SAS with treatment used as a fixed effect and initial body weight used as a covariate. The main effect of diet did not differ (P ≥ 0.127) for any of the tissues. However, E₂ treatment differed for the endometrium (P ≤ 0.001), but not for the ovary or hypothalamus (P ≥ 0.528). In the endometrium, ERα was upregulated (P ≤ 0.024) in ACC heifers (8.44 ± 1.21) as compared with ACCEST (2.69 ± 1.43), and CONEST heifers (2.63 ± 1.22). Gene ERβ was also upregulated more (P ≤ 0.052) in ACC heifers (4.16 ± 0.52) than ACCEST (2.11 ± 0.61) or CONEST heifers (1.46 ± 0.52) in the endometrium. Additionally, ERβ was upregulated (P < 0.001) in CON heifers (4.56 ± 0.52) as compared with CONEST heifers, and tended (P = 0.071) to be upregulated in ACCEST heifers. Overall, results indicated that diet alone did not affect expression of ERα or ERβ in reproductive tissues of pre-pubertal heifers. However, treatment with E₂ in pre-pubertal heifers affected relative gene expression of key genes within the hypothalamic-gonadal pathway. Determining the effect of estrogen on the development of reproductive tissues is key to understanding signaling pathways that activate the onset of puberty.