Simple SummaryOvarian cancer (OC) comprises a heterogeneous group of hormone-dependent diseases with very high mortality. Estrogens have been shown to promote the progression of OC; however, their exact role in OC subtypes remains unknown. Here, we investigated the local estrogen biosynthesis in OC. We performed targeted transcriptomics and estrogen metabolism analyses in high-grade serous OC (HGSOC) cell lines that differed in chemoresistance status and compared these data with publicly available transcriptome and proteome data for HGSOC tissues. In HGSOC cells, estrogen metabolism decreased with increasing chemoresistance. In highly chemoresistant cells and platinum-resistant HGSOC tissues, HSD17B14 expression was increased. Proteome data showed differential levels of HSD17B10, SULT1E1, CYP1B1, and NQO1 between the four HGSOC subtypes. Our results confirm that estrogen biosynthesis differs between different HGSOC cell models and possibly between different HGSOC subtypes. Such differentially expressed enzymes have potential as targets in the search of new treatment options.Ovarian cancer (OC) is highly lethal and heterogeneous. Several hormones are involved in OC etiology including estrogens; however, their role in OC is not completely understood. Here, we performed targeted transcriptomics and estrogen metabolism analyses in high-grade serous OC (HGSOC), OVSAHO, Kuramochi, COV632, and immortalized normal ovarian epithelial HIO-80 cells. We compared these data with public transcriptome and proteome data for the HGSOC tissues. In all model systems, high steroid sulfatase expression and weak/undetected aromatase (CYP19A1) expression indicated the formation of estrogens from the precursor estrone-sulfate (E1-S). In OC cells, the metabolism of E1-S to estradiol was the highest in OVSAHO, followed by Kuramochi and COV362 cells, and decreased with increasing chemoresistance. In addition, higher HSD17B14 and CYP1A2 expressions were observed in highly chemoresistant COV362 cells and platinum-resistant tissues compared to those in HIO-80 cells and platinum-sensitive tissues. The HGSOC cell models differed in HSD17B10, CYP1B1, and NQO1 expression. Proteomic data also showed different levels of HSD17B10, CYP1B1, NQO1, and SULT1E1 between the four HGSOC subtypes. These results suggest that different HGSOC subtypes form different levels of estrogens and their metabolites and that the estrogen-biosynthesis-associated targets should be further studied for the development of personalized treatment.