Due to the immunogenic differences in the FSH-β subunits among animal species and the need for preparing specific gonadotropic hormones specific for buffaloes, the present work was aimed to prepare recombinant buffalo follicle stimulating hormone for using in improving the fertility of Egyptian buffaloes. Production of recombinant buffalo follicle stimulating hormone (rbuFSH) in E.coli by using recent biotechnological tools .Buffalo anterior pituitaries were collected from slaughtered buffaloes ,the buffalo common α-subunit and FSH β-subunit cDNAs was cloned and expressed to produce recombinant FSH from buffalo species in vitro. The RNAs extracted from buffalo pituitary glands will be reverse-transcribed and amplified by PCR and RT-PCR. The cDNAs corresponding to both subunits of buffalo hormones was cloned into the expression vector E.coli cells (DH5α) and pGEMT-Easy vector and transfected into pET 28a, pET 15b cells or pCR2.1-TOPO vector. Subcloning of the different subunits was carried out in pET vectors. Expression of genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant buffalo FSH secreted in culture media was characterized by an in vitro bioassay. The recombinant products were injected to immature female rats compared to PMSG(standard) to stimulate ovarian growth and inducing of multiple follicles. The results revealed that the possibility of producing recombinant buff. FSH in vitro which are possessing efficient biological activity,stimulated the ovarian activity of immature female rats compared with standard PMSG hormone , which can be applied in buffalo-cows for improvement of reproductive efficiency .