Background: Dehydroepiandrosterone sulfate (DHEAS) is metabolized to active androgens and estrogens, which may have a role in the development of endometrial cancer. Methods: We studied DHEAS conversion to dehydroepiandrosterone (DHEA) in normal and neoplastic endometrium utilizing gas chromatography-mass spectral (GC-MS) analysis. Endometrial homogenate was incubated with known amounts of DHEAS for 4 h at 37°C. Methanol extract was separated from debris by centrifugation, concentrated to 200 μl and 1 μl injected into the GC-MS instrument, equipped with a CP-Sil 8 column. DHEAS and DHEA areas were calculated by autoquantization and DHEA/DHEAS ratio was used for comparing sulfatase activity among normal endometrium (n = 6), Stage I endometrioid carcinoma (EC) (n = 15), Stage I mixed mesodermal Müllerian tumor (MMMT) (n = 6) and Stage I uterine papillary serous carcinoma (UPSC) (n = 7). Results: DHEA/DHEAS ratios in normal endometrium, EC, MMMT and UPSC were 1.45 ± 1.10, 5.63 ± 3.27, 2.88 ± 0.99, and 3.04 ± 1.76, respectively. Sulfatase activity was significantly higher in EC when compared with normal endometrium (p < 0.001), MMMT (p < 0.05), and UPSC (p < 0.05). The enzyme activity did not differ significantly between low-grade and high-grade EC tumors (5.8 ± 2.77 and 5.49 ± 3.84, respectively, p > 0.05). Conclusion: Stage I EC have higher sulfatase activity than normal endometrium, and Stage I MMMT and UPSC tumors.