The effects of CoCl2 on retinoic acid (RA)-treated embryoid bodies (EBs) were investigated. Four-day EBs were treated with 5 x 10(-6) of RA for 4 d, then subjected to attached culturing for 7 d in the presence of CoCl2 at 0, 20, and 100 mu M. Differentiation into MAP2-and GFAP-immunopositive cells was inhibited by CoCl2 in a dose-dependent manner. Next, RA-treated EBs were dissociated into single cells and cultured for 7 d at an initial cell density of 1 x 10(3)/cm(2). The number of cells increased in a CoCl2-dose dependent fashion. In cultures with 100 PM of CoCl2, more than 90% of the cells were immunopositive for nestin and nestin-immunopositive cells formed clusters, while there were few cells immunopositive for MAP2 or GFAP. These results suggest that CoCl2 inhibits neural differentiation of RA-treated EB cells and promotes the proliferation of nestin-immunopositive cells, i.e., embryonic stem (ES)-derived neural stem-like cells.