Human embryonic stem (ES) cells have been established either from fresh or frozen embryos. The recovery rates of undifferentiated human ES cells after cryopreservation with conventional slow-rate freezing and rapid-thawing methods are relatively low. The purpose of this study was to improve cryopreservation efficiency by modifying conventional methods with addition of trehalose. Immature oocytes donated from patients undergoing IVF treatment were utilized to generate blastocysts. One human ES cell line (named hES1) was established and characterized in detail. The hES1 cells expressed regular human ES cell markers, including stage-specific embryonic antigens SSEA-3, SSEA-4, tumour rejection antigens TRA-1-60, TRA-1-81 and octamer-binding transcription factor Oct-4 with high levels of alkaline phosphatase and telomerase activities. Cells could be differentiated to form teratomas in vivo. With slow-rate freezing and rapid-thawing methods modified by adding trehalose, the recovery rate of undifferentiated hES1 cells has been greatly improved from 15 to 48%. Cells retained pluripotency with normal karyotype after thawing. The results indicated that the use of trehalose is efficient and convenient for cryopreservation of human ES cells.