The bovine trophoblast produces significant amounts of estrogens. In maternal and fetal blood, estrogens occur predominantly in sulfonated forms, which are unable to bind to estrogen receptors (ESRs). However, estrogens may act as local factors in ESR-positive trophoblast cells or in the adjacent caruncular epithelium, which in addition to ESR highly expresses steroid sulfatase. Estrogen sulfonation is catalyzed by the cytosolic enzyme SULT1E1. Previous studies clearly indicated the trophoblast as the primary site of estrogen sulfonation. However, investigations into the cellular localization of SULT1E1 yielded conflicting results. In situ hybridization studies detected SULT1E1 mRNA only in trophoblast giant cells (TGCs), whereas in immunohistochemical experiments the SULT1E1 protein was virtually restricted to uninucleated trophoblast cells (UTCs). The aim of this work was to resolve this conflict by analyzing SULT1E1 expression in isolated UTCs and TGCs. Highly enriched pools of UTCs and TGCs were obtained from four bovine placentas (Days 118–130 of gestation) using an optimized fluorescence-activated cell sorting procedure. UTC and TGC pools were analyzed by quantitative RT-PCR and Western blot experiments to measure the amounts of SULT1E1 transcript and protein, respectively. In contrast to previously published results, both SULT1E1 transcript and SULT1E1 protein were clearly present in the UTC and TGC pools. However, some evidence indicated a higher transcript concentration in TGCs and a higher amount of protein in UTCs. Thus, our results resolve the conflicting results on the localization of SULT1E1 from earlier studies and suggest that posttranscriptional mechanisms play an important role in the control of SULT1E1 expression during TGC differentiation.