In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus-oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5-5.5% CO2 under various conditions (37-39 °C and 5-20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.