Aims:  There is increasing concern about the effect of environmental toxins and endocrine disrupters on human spermatogenesis but ethical considerations prohibit direct research. Therefore, we developed a method of isolating mature pig Sertoli cells and studying the levels of mRNA of transferrin and inhibin, markers of Sertoli cell function, using real-time polymerase chain reaction analysis. Methods:  Sertoli cells from mature Meishan boars were isolated and cultured. The mRNA was isolated from the cells after 3 days of treatment with follicle-stimulating hormone (FSH), testosterone and β-estradiol. The amounts of transferrin and inhibin mRNA were analyzed by real-time polymerase chain reaction and the relative level of mRNA was calculated. Results:  FSH tended to increase the levels of transferrin and inhibin mRNA, as did the combinations of FSH and the steroids. The effect of testosterone and β-estradiol alone on the level of mRNA was less than that of FSH. Beta-estradiol treatment resulted in a dose-dependent decrease in mRNA. Conclusion:  The Sertoli cell culture used in the present study appears to have a normal secretory function because the mRNA levels of both markers increased after FSH treatment. The suppressive effect of β-estradiol on inhibin mRNA level suggests that β-estradiol has an effect on the function of Sertoli cells. (Reprod Med Biol 2005; 4: 259 -264).