Glutathione S-transferase M1 (GSTM1) enzyme serves as a steroid-binding protein by its ability to bind to testosterone and estradiol. The levels of total estradiol and testosterone were measured by using an electrochemiluminescence immunoassay in serum and seminal plasma from 103 subjects including 62 subfertile patients. GSTM1 polymorphism was examined using polymerase chain reaction. The estradiol and testosterone levels in seminal plasma were not different in control and subfertile subjects. No role for GSTM1 enzyme as a steroid-binding protein seemed likely as there was also no significant difference in seminal plasma estradiol and testosterone levels according to GSTM1 genotype. Significant positive correlations were found between seminal estradiol and serum estradiol in infertile males, and between seminal testosterone and serum testosterone in fertile males, independent of GSTM1 genotype. GSTM1 polymorphism is not a genetic risk factor of seminal estradiol and testosterone levels in infertile males although further studies are warranted.