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Sertoli cells metabolize glucose, converting it to lactate that is used by developing germ cells for their energy metabolism. Androgens and oestrogens have metabolic roles that reach far beyond reproductive processes. So, the main purpose of this study was to examine the effect of sex steroid hormones on metabolite secretion/consumption in human Sertoli cells. Human Sertoli cell‐enriched primary cultures were maintained in a defined medium for 50 h and glucose, pyruvate, lactate and alanine variations were determined using ¹H‐NMR spectra analysis, in the absence or presence of 100 nm 17β‐estradiol (E₂) or 100 nm 5α‐dihydrotestosterone (DHT). The mRNA expression levels of glucose transporters, lactate dehydrogenase and monocarboxylate transporters were also determined using semi‐quantitative RT‐PCR. Cells cultured in the absence (control) or presence of E₂ consumed the same amounts of …