Glial cell line-derived neurotropic factor (GDNF) was first characterized by its actions on central nervous system neurons. GDNF messenger RNA was expressed in many peripheral tissues in addition to brains. We demonstrated that GDNF had a marked stimulatory effect on the proliferation of Sertoli cells during the early postnatal period of rat testis development. The action of GDNF was examined in in vitro organ culture using testicular fragments from 6-day-old rats. [3H]Thymidine incorporation into testicular fragments cultured for 3 days was significantly stimulated by GDNF in the presence of FSH. Neither LH nor testosterone had such synergism with GDNF. The stimulation was GDNF dose and [3H]thymidine exposure time dependent and was specifically inhibited by both anti-GDNF and anti-GDNF receptor (RET) antibodies. Immunohistological section labeled with 5'-bromo-2'-deoxyuridine at the end of in vitro culture demonstrated a dramatic increase in both total and labeled Sertoli cells after combined treatment with FSH and GDNF. These findings suggest that GDNF might play an important role in regulation of Sertoli cell number as a local factor in early postnatal period of rat testis development.