The origin of the extraembryonic ectoderm of the chorion in the mouse embryo has long been the source of some controversy. Various manipulative studies suggested that it arose from the trophectoderm and not the inner cell mass (ICM) of the blastocyst. However, recent studies on the development of isolated ICMs in vitro have reported the formation of tissues morphologically resembling extraembryonic ectoderm. One explanation not excluded by previous studies is that the chorionic ectoderm is of dual origin, from both ICM and trophectoderm. The present study provides a more detailed analysis than previously possible of the in vivo fate of ICMs in chimeras, using a sensitive assay for glucose phosphate isomerase (GPI) isozymes which permits study of the chorionic ectoderm alone. In a large series of blastocyst injection chimeras, no donor ICM contribution to the mature chorionic ectoderm could be detected, donor activity appearing only in the embryonic fraction. Thus, the in vitro results cannot be readily explained by dual origin of the chorionic ectoderm and remain in conflict with existing in vivo data. Analysis of most ICM/morula chimeras revealed the same pattern, but a few showed ICM contributions to the trophoblast fractions, suggesting that some ICM cells retain the potential to form trophectoderm derivatives in vivo. © 1979.