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Abstract Context The testis produces hormones controlling sex differentiation, development, and reproduction. Their quantification in physiological conditions can aid our understanding of the endocrine function of the testis. Objective To measure the concentrations of steroid hormones and FSH, LH, inhibin B, anti-Müllerian hormone (AMH), and insulin-like factor 3 (INSL3) in the testicular and femoral veins, femoral artery, and seminal fluid in healthy men. Design Sample collection was made by angiographic catheterization under fluoroscopy from the testicular and femoral veins. Femoral artery samples were collected via needle puncture and seminal fluid was isolated from semen samples. Hormones were measured via liquid chromatography mass spectrometry and immunoassays. Participants Seven volunteers (aged 22-49 years) without self-reported diseases and normal semen quality were included. Main outcomes Concentrations of steroid hormones, FSH, LH, inhibin B, AMH, and INSL3 in the testicular vein, femoral artery, femoral vein, and seminal fluid. Results Expectedly, testosterone was 66-fold higher in the testicular vein compared to the femoral artery, but surprisingly, levels of 11-deoxycortisol and 11-deoxycorticosterone were 4- and 20-fold higher in the testicular vein compared to the femoral artery. In line with their production site in the testis, INSL3 and inhibin B were 200- and 2-fold higher in the testicular vein compared to the femoral artery. Unexpectedly, concentration of AMH was not different between the testicular vein and femoral artery despite its documented production in the testis. Conclusion and relevance Our results provide insight into the endocrine function of the testis under physiological conditions and constitute a reference for further research in male reproductive function.