Embryonic stem (ES)-like cells were isolated from in vivo-produced cat embryos. Total of 101 blastocysts were collected from female cats. The inner cell mass (ICM) were mechanically isolated and cultured on mitomycin-C-treated cat embryonic fibroblast feeder layers in medium supplemented with knockouttrade mark Serum Replacement (KSR-medium) or fetal bovine serum (FBS-medium). Putative ES-like cell colonies developed in both KSR- and FBS-medium conditions, but formed domed and flat colonies, respectively. ICM cell attachment and ES-like cell colony formation were significantly higher in KSR-medium, but subsequent cell proliferation was significantly lower than in FBS-medium. For passaging, 32 and 18 colonies in KSR- and FBS-medium were separated by enzymatic dissociation or mechanical disaggregation. Enzymatic dissociation resulted in cell differentiation; however, mechanical disaggregation generated cells that remained undifferentiated over more than four passages and yielded two cat ES-like cell lines that continued to grow for up to eight passages in FBS-medium. These cells had typical stem cell morphology, expressed high levels of alkaline phosphatase activity, and were positive for the ES cell-markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, and SSEA-4. These cells formed embryoid bodies (EBs) in suspension culture after extended suspension culture. When simple EBs were cultured on tissue culture plates, they differentiated into several cell types, including epithelium-like and neuron-like cells. In addition, EBs were positive for mesoderm marker, desmin. After prolonged in vitro culture, some colonies spontaneously differentiated into beating myocardiocytes, and were positive for alpha-actinin. These observations indicate that cat ES-like cells were successfully isolated and characterized from in vivo-produced blastocysts.