Abstract Purpose: The concentration of estradiol (E2) in breast tumors is significantly higher than that in plasma, particularly in postmenopausal women. The contribution of local E2 synthesis versus uptake of E2 from the circulation is controversial. Our aim was to identify possible determinants of intratumoral E2 levels in breast cancer patients. Experimental Design: The expression of genes involved in estrogen synthesis, metabolism, and signaling was measured in 34 matched samples of breast tumor and normal breast tissue, and their correlation with estrogen concentrations assessed. Results: ESR1 (9.1-fold; P < 0.001) and HSD17B7 (3.5-fold; P < 0.001) were upregulated in ER+ tumors compared with normal tissues, whereas STS (0.34-fold; P < 0.001) and HSD17B5 (0.23-fold; P < 0.001) were downregulated. Intratumoral E2 levels showed a strong positive correlation with ESR1 expression in all patients (Spearman r = 0.55, P < 0.001) and among the subgroups of postmenopausal (r = 0.76, P < 0.001; n = 23) and postmenopausal ER+ patients (r = 0.59, P = 0.013; n = 17). HSD17B7 expression showed a significant positive correlation (r = 0.59, P < 0.001) whereas HSD17B2 (r = −0.46, P = 0.0057) and HSD17B12 (r = −0.45, P = 0.0076) showed significant negative correlations with intratumoral E2 in all patients. Intratumoral E2 revealed no correlation to CYP19, STS, and HSD17B1 expression. Multivariate models comprising ESR1 and plasma E2 predicted between 50% and 70% of intratumoral E2 variability. Conclusion: Uptake due to binding to the ER, rather than intratumoral estrogen synthesis by aromatase or sulfatase, is the single most important correlate and a probable determinant of intratumoral E2. An increased expression of HSD17B7 may explain the increased ratio of E2 to estrone (E1) in breast tumors compared with normal tissue. Clin Cancer Res; 16(6); 1790–801