The adult human Sertoli cells produced lactate, estradiol-17beta, transferrin and inhibin; germ cells modulate synthesis of these compounds. In order to study the functional features of human Sertoli cells in vitro, the aim of this study was to measure the lactic dehydrogenase (LDH), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP) and creatine kinase activities (CK) in primary cultures of Sertoli cells prepared from young men (mean age: 29 years, n = 11). Five LDH isozymes have been found in Sertoli cells, the main fractions being the LDH3 and LDH4; each of them represented 30% of the total LDH activity. Furthermore, CK and ALP activities were measured in Sertoli cells. It is of note that the Sertoli cell ALP activity was 50% lower than that of germ cells. Whatever the Sertoli cell parameter measured herein, there is a great variability between patients and FSH (dbc AMP, as well as retinol, insulin, testosterone) is poorly effective in improving these enzymatic activities in vitro. We have confirmed that GGT was exclusively present in Sertoli cells and thus may be considered as a specific marker. LDH is involved in Sertoli cell glucose transformation and thus provided energetic substrates for germ cells. In contrast, the roles of CK and ALP remains to be clarified. In conclusion, we have demonstrated the existence of several enzymes namely LDH, GGT, ALP and CK in Sertoli cells prepared from adult human testis.