Yes-associated protein (YAP) is an evolutionarily conserved protein that regulates organ size, but is not yet reported on in dairy cattle. The hypothesis was that in vivo estradiol administration increases the abundance and nuclear localization of YAP in prepubertal bovine mammary epithelial cell (MEC) and myoepithelial (MYO) cells. Twelve Holstein heifers (6 ± 2 d old) received enhanced milk replacer (EH; 28.9% CP, 26.2% fat, DM basis; 1.08 kg powder DM/d), and were offered starter (26% CP; 23.2 g DM/kg BW from 4 wk of age). Heifers received an ear implant containing either estradiol EH-E₂ (n = 6) (25.7 mg estradiol) or placebo EH (n = 6) at weaning (8 wk). Post weaning, both groups received starter (25.6% CP, 4.0% fat, 19.8% neutral detergent fiber; DM basis). Heifers were slaughtered at 10 wk. Mammary parenchyma was collected (EH-E₂, n = 6; EH, n = 4) and immunofluorescently labeled for YAP, MYO nuclei, and MEC nuclei. Images, manually identified compartments, and nuclear objects were manipulated with CellProfiler. Data were analyzed using SAS 9.4 with the fixed effect of treatment and random effect of heifer within treatment. Generally, YAP was localized to the cytoplasm of MEC and nuclei of MYO. Within MEC, estradiol treatment did not affect nuclear (P = 0.974), cytoplasmic (P = 0.394), or total intensity of YAP (P = 0.979). Similarly, within MYO, estradiol treatment did not affect nuclear (P = 0.997), cytoplasmic (P = 0.624), or total intensity of YAP (P = 0.308). In this experiment, in vivo estradiol administration did not affect the sub-cellular localization of YAP (nucleus versus cytoplasm) nor the intensity (abundance) of YAP within MEC and MYO compartments. Findings suggest: 1) sub-cellular YAP localization is compartmentalized in bovine mammary tissue and may affect growth independent of estradiol, 2) YAP signaling might work in concert with non-estradiol pathways to affect organ size, as estradiol treatment 2 wk before slaughter did not elicit change in YAP localization in MEC and MYO.