Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slow-rate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.