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The normal rate of blastocysts produced through IVF ranges from 8–10%. For increasing the number of blastocysts in buffalo one possible way could be to use the parthenogenetically activated oocytes after in vitro maturation. The process by which an oocyte can develop into an embryo in the absence of sperm is known as parthenogenesis. From the parthenotes one can derive the embryonic stem cell-like cells. Current knowledge on the biology of mammalian embryonic stem cell-like cells (ESC) is stunningly sparse in light of their potential value in studies of development and functional genomics as well as to salvage the endangered species. Keeping this in view present study was undertaken to isolate inner cell mass (ICM) from in vitro produced buffalo embryos through parthenogenetic activation (parthenotes) for production and culturing of ES cell-like cells. Buffalo embryos were produced from the oocytes which were activated by the treatment of calcium ionophore (A23187) followed by 6-DMAP. The presumptive embryos produced were subjected to in vitro culture (IVC). A total of 80 blastocysts (20 early blastocysts, 16 expanded blastocysts and 44 hatched blastocysts) were used for ES cell- like cells isolation. ICM was isolated by mechanical / enzymatic treatment (with 0.25%Trypsin–EDTA), washed and transferred onto mitomycin-C (10 μl/ml) treated feeder layers of buffalo fetal fibroblasts. Culture medium for the isolated cells consisted of 80% DMEM supplemented with 20% fetal bovine serum, 1000U/ml of Leukemia inhibitory factor (LIF), 1% non-essential amino acids and 0.1 mM betamercaptoethanol. After 2–3 days, ICM-derived primary colonies of buffalo embryonic stem cell-like cells were dissociated mechanically and subcultured on mitomycin–C treated feeder layer in fresh medium. To passage the putative ES cell-like cells, morphologically undifferentiated colonies were selected individually and sub-cultured in fresh medium. Buffalo ES cell-like cell lines (bES1) derived from ICM passed eight sub cultures (passages). However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology and expressed specific markers such as alkaline phosphatase and Oct-4. In conclusion, buffalo embryos produced in vitro from parthenogenetically activated oocytes could be used as a source for the production of embryonic stem cell-like cells. (poster)