Pluripotent embryonic stem (ES) cells isolated from inner cell mass (ICM) of blastocyst-stage embryos are capable of differentiating into various cell lineages and demonstrate germ-line transmission in experimentally produced chimeras. These cells have a great potential as tools for transgenic animal production, screening of newly-developed drugs, and cell therapy. Miniature pigs, selectively bred pigs for small size, offer several advantages over large breed pigs in biomedical research including human disease model and xenotransplantation. In the present study, factors affecting primary culture of somatic cell nuclear transfer blastocysts from miniature pigs for isolation of ES cells were investigated. Formation of primary colonies occurred only on STO cells in human ES medium. In contrast, no ICM outgrowth was observed on mouse embryonic fibroblasts (MEF) in porcine ES medium. Plating intact blastocysts and isolated ICM resulted in comparable attachment on feeder layer and primary colony formation. After subculture of ES-like colonies, two putative ES cell lines were isolated. Colonies of putative ES cells morphologically resembled murine ES cells. These cells were maintained in culture up to three passages, but lost by spontaneous differentiation. The present study demonstrates factors involved in the early stage of nuclear transfer ES cell isolation in miniature pigs. However, long-term maintenance and characterization of nuclear transfer ES cells in miniature pigs are remained to be done in further studies.