An embryonic stem (ES)-like cell culture system RESC from a commercially important freshwater carp, Labeo rohita, was developed using blastula stage embryos. The cells were cultured in Leibovitz-15 (L-15) medium in gelatin-coated cell culture flask supplemented with 15 % fetal bovine serum along with 10 ng ml(-1) basic fibroblast growth factor at 28 °C under feeder-free conditions. The ES-like cells were characterized by their unique morphology, alkaline phosphatase activity, embryoid body formation tendency, expression of transcription factor Oct4, and consistent chromosome count. The RESC cells when treated with retinoic acid differentiated into cells of different lineages. The RESC developed from mid-blastula embryos of L. rohita would be a useful tool for cellular differentiation and gene expression studies.