The distribution of a blood group I-associated antigen in the postimplantation mouse embryo was examined using immunohistochemical, immunofluorescence, and immunoblotting techniques. The following results were obtained: 1. The I antigen defined by monoclonal antibody C6 was largely, but not entirely, restricted to mesodermal cells and their derivatives in day-6.5 to day-10 embryos. A gradual increase in the surface expression of the I antigen was observed as mesodermal cells moved away from the primitive streak in posterior and lateral directions between the embryonic ectoderm and visceral endoderm. I expression declined in hindlimb-bud-stage embryos in a posterior-to-anterior progression, and was not observed in fetal tissues after day 11. 2. The appearance of mesodermal cells was associated with a switch in surface carbohydrate expression. Two-color labeling experiments using SSEA-1 and C6 monoclonal antibodies suggested that mesodermal cells leaving the primitive streak lose surface Lex and acquire I. This suggestion was supported by flow-cytofluorimetric analyses of single cell preparations of day-8 and day-10 embryos: cell-surface Lex expression declined markedly between days 8 and 10, while I expression increased. 3. The glycoproteins carrying Lex and I antigens were observed to change in molecular weight during early development. Lex was identified on a broad glycoprotein band with an apparent molecular weight greater than 200,000, whereas the I antigen was identified on glycoprotein bands with apparent molecular weights of 155,000 (day 8), and 140,000, 155,000, 210,000, and 250,000 (day 10). The results indicate that the I antigen is a useful marker of early mesoderm in the mouse embryo.