Insulin-like factor 3 (INSL3) serum levels were measured in 135 andrologically well-characterized normal men and 85 patients with testicular disorders to investigate how the hormone, which is a major secretory product of human Leydig cells, is related to testosterone (T), LH, and semen quality. INSL3 was measured by using a newly developed fluorescence immunoassay. Median (2.5-97.5 percentiles) INSL3 serum levels were as follows: normal men (n = 135), 0.99 (0.55-1.73) ng/ml; infertile men (n = 23), 1.11 (0.60-2.07) ng/ml; anorchid men (n = 21), nondetectable (ND); patients with 47, XXY, Klinefelter syndrome (n = 21), 0.12 (ND-0.78) ng/ml; men with hypogonadotropic hypogonadism and T substitution (n = 11), ND; and men with hypogonadotropic hypogonadism and human chorionic gonadotropin (hCG) treatment (n = 5), 0.36 (0.13-0.73) ng/ml. Before testicular biopsy, two infertile men had blood samples drawn directly from vena spermatica. Here, the serum INSL3 levels were 15-fold higher than in serum from peripheral blood samples (13.84 and 14.00 ng/ml, respectively). In two unilaterally orchiectomized former testis cancer patients, who underwent hCG stimulation test, INSL3 serum levels were unchanged 72 and 96 h after hCG stimulation. In conclusion, we provide a normal range for INSL3 serum levels in adult men and show that the majority, if not all, circulating INSL3 derives from the testes. Furthermore, our data strongly indicate that INSL3 secretion is dependent on the differentiating effect of LH on Leydig cells but independent of the steroidogenic LH-mediated action. Thus, even though T and INSL3 are both dependent on LH, these two Leydig cell hormones are regulated differently.