The present study was designed to examine the effect of different doses of FSH on the growth, steroidogenesis and DNA synthesis of two different sizes of buffalo ovarian preantral follicles (PFs) during in vitro culture. Buffalo ovaries were collected from a local abattoir and PFs of small (<150 µm) and large (>150 µm) sizes were isolated through the microdissection method and cultured in TCM-199 supplemented with 10% FBS, 1% ITS and 30 ng/ml EGF (control group) at 38.5°C in 5% CO2 with maximum humidity. The addition of three different doses of FSH (0.5, 1.0 and 2.0 µg/ml) in the control medium served as the treatment groups. The culture medium was replenished every third day and spent culture medium was stored at -30°C for steroid hormone analysis. The results showed that the supplementation of FSH in the culture medium stimulated (P < 0.05) follicular growth, steroidogenesis and DNA synthesis compared to the control. The addition of FSH at 1.0 µg/ml in the culture medium stimulated better (P < 0.05) follicular growth, DNA synthesis and steroidogenesis compared to 0.5 µg/ml FSH, whereas a higher dose of FSH (2.0 µg/ml) inhibited follicular growth in both sizes of follicles. In conclusion, the present study demonstrated that FSH plays a key role during the development of buffalo ovarian preantral follicles, as it stimulated in vitro survival, growth rate, steroidogenesis and DNA synthesis of buffalo preantral follicles at an optimal dose of 1.0 µg/ml.