We examined the expression of cell-surface markers on subpopulations of mouse embryonic stem (ES) cells to identify those that were associated with cells that had the highest pluripotency. Flow cytometry analysis revealed a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM-1) and stage-specific embryonic antigen (SSEA)-1 in ES cells. Almost all SSEA-1+ cells expressed a high level of PECAM- 1, and reversible repopulation was observed between PECAM- 1+SSEA-1+ and PECAM-1+SSEA-1- cells. The ES cells carrying the lacZ gene were sorted into three subpopulations: PECAM- 1-SSEA-1-, PECAM-1+SSEA-1-, and PECAM-1+SSEA-1+. Quantitative reverse transcription-polymerase chain reaction revealed a low level of Oct3/4 mRNA expression and an elevation in differentiation maker gene expression in PECAM-1- cells. To compare the pluripotency of these three subpopulations, a single cell from each was injected into eight-cell embryo and ES cells identified at later stages by X-gal staining. At the blastocyst stage, PECAM-1+ SSEA-1+/- cells were found to have differentiated into epiblast cells in high numbers. In contrast, PECAM- 1- cell derivatives localized in the primitive endoderm or trophectoderm. At 6.0-7.0 days post coitum, many PECAM-1+SSEA- 1+ cells were found in the epiblast, but few beta-gal+ cells were detected in any regions of embryos that were injected with cells from the other two populations. These results showed that the expression levels of PECAM-1 and SSEA-1 in ES cells correlated closely with their pluripotency and/or their ability to incorporate into the epiblast of chimeric embryos.