We have isolated three embryonic stem (ES) cell lines from C3H/He mice using mouse STO cells as a feeder layer. One ES cell line (H-1) was male, and two (H-2 and H-3) were female, as determined by polymerase chain reaction, in situ hybridization, and karyotype analyses. All were immunocytochemically reactive with a C3H strain-specific antibody. Injection of cells from the female ES H-3 line into C57BL/6 blastocysts yielded four chimeras with slight coat color chimerism. All chimeras were male, and as expected, no germline-transmission was observed. By contrast, when male ES H-1 cells were injected into the perivitelline space of 8-cell C57BL/6 embryos, one male mouse with overt coat color chimerism was recovered, and it produced ES H-1-derived offspring exclusively. This germline-transmissible C3H/He cell line represents a novel addition to those ES lines currently employed for gene manipulation studies of development.