Congenital adrenal hyperplasia (CAH) is a recessive condition that affects the adrenalglands. Despite life-long replacement therapy with glucocorticoids and mineralocorticoids,adult patients with CAH often experience impaired gonadal function. In pubertal boys andin men with CAH, circulating testosterone is produced by the adrenal glands as well as thetesticular, steroidogenic cells. In this European two-center study, we evaluated the functionof Leydig and Sertoli cells in 61 boys and men with CAH, primarily due to 21-hydroxylasedeficiency. Despite conventional hormone replacement therapy, our results indicated asignificant reduction in serum concentrations of both Leydig cell-derived hormones (i.e.insulin-like factor 3 (INSL3) and testosterone) and Sertoli cell-derived hormones (i.e. inhibinB and anti-Müllerian hormone) in adult males with CAH. Serum concentrations of INSL3were particularly reduced in those with testicular adrenal rest tumors. To our knowledge,this is the first study to evaluate circulating INSL3 as a candidate biomarker to monitorLeydig cell function in patients with CAH.