The aim of this study was to examine the effects of 22R-hydroxycholesterol (22R-HC), folliclestimulating hormone (FSH) and luteinizing hormone (LH) on estradiol and progesterone production by cat granulosa cells. Granulosa cells from follicles were collected and cultured for up to 5 days in 24 well plates containing Dulbecco’s Modified Eagle’s Medium (DMEM)/HAM F-12 supplemented with 10-7 M androstenedione, 0.1% ITS premix and 0.1% bovine serum albumin, in the presence or absence of 22R-HC (10 µg/ml), FSH or LH (10, 100 ng/ml each) on first and third day. Additionally, 5% fetal calf serum was added into the culture medium for the first 24 h. Treatment of cells with 22R-HC resulted in an increase (P < 0.05) in progesterone and estradiol production on days 3 and 5 of the culture. Incubation of cells with FSH (10 and 100 ng/ml) resulted in significant stimulations of progesterone (P < 0.001) whilst incubation had no effect on estradiol production. None of the LH doses (10 and 100 ng/ml) had any effect on progesterone production by granulosa cells during the culture time. With the inclusion of 22R-HC into the culture system, progesterone synthesis was enhanced (P < 0.001) in the presence of all FSH doses.