Follicle and oocyte growth is under the control of systemic gonadotropins and various local intraovarian factors. The present study was undertaken to examine the efficacy of peptide(s) isolated from ovine follicular fluid on preantral and antral follicles, oocytes, cumulus and granulosa cells, and oviductal epithelial cell growth in vitro in a heterologous species (Bubalus bubalis). Follicular fluid aspirated from different follicles sizes of slaughterhouse-derived ovine ovaries was made cell free by centrifugation and steroid free by charcoal treatment. Follicular fluid was fractionated using ammonium sulphate precipitation at various saturation levels and then subjected to gel filtration chromatography. The 35-50% fraction yielded a detectable peak and a peptide of 30.1 kDa as examined by SDS-PAGE under reducing conditions. The isolated ovine follicular fluid peptide was tested for effects at different doses on in vitro preantral and antral follicle growth, cumulus cell expansion, in vitro oocyte maturation, and changes in protein, calcium, and phosphorus levels of oocytes after culture and somatic (granulosa, cumulus, and oviductal epithelial) cell monolayer formation in buffalo. The isolated peptide inhibited oocyte maturation (31% vs. 80%) and antral follicle growth (6.5 µm/day vs. 1.4 µm/day) at 1 µg/ml and 0.5 µg/ml levels, respectively, compared to control but had no effect on growth of somatic cells and preantral follicles in vitro. The protein, calcium, and phosphorus contents of oocytes were found to decrease in oocytes cultured in maturation medium containing the isolated peptide. The present study demonstrated the effect of an ovine intrafollicular factor regulating oocyte and antral follicle development in buffalo