In producing mutant mice by gene-targeting and gene-trapping in embryonic stem (ES) cells, the efficient colonization of the mutant ES cells into germline is still a critical matter. We have established a new line of ES cells, TT2, from an F1 embryo between a C57BL/6 female and a CBA male. When the TT2 cells were injected into blastocysts, the colonization into each tissue was very low. However, when injected into eight-cell embryos, the cells segregated inside the blastomeres, localized in an inner cell mass of blastocysts developed 1 day later, and colonized efficiently in each tissue of the pups. The pups were disproportionately male, about half of which were composed of TT2-derived cells primarily; in more than 70% of the males, TT2-derived cells were dominant, accounting for over half of the total cells. When these males were mated, they exclusively yielded TT2-derived offspring. The germline-differentiating potency was stable during 3 weeks of culture. Twenty-one of 24 mutant clones independently isolated yielded germline chimeras, and 19 clones yielded them in a rate comparable to that of the parent cells. Thus, TT2 cells can serve as a valuable vehicle for the production of mutant mice.