The division of single cells, isolated from an 8-cell mouse embryo, to give 2 x 1/16 cells has been studied by sampling cells for analysis at defined stages during and after the division. Cells were analyzed for evidence of polarity in their surface organization as assessed by fluorescent ligand binding and distribution of microvilli. Individual 1/8 cells are polarized. At division, most (82%) divide such that both the pole of ligand binding and the pole of microvilli are distributed to only one of the two daughter cells. A couplet is thereby formed with a large polar cell and a small apolar cell. Some 1/8 cells divide through the pole, generating a couplet of two polar cells, the poles being contiguous at the midbody. Elements of the surface polarity observed in the 1/8 cells can be found at all stages throughout division. Analysis of couplets of cells derived from newly formed 16-cell morulae also reveals that most consist of a polar:apolar pair and some consist of a polar:polar couplet in which the poles are contiguous at the midbody. The results indicate that two distinct cell populations are generated at division. These cells are known to occupy different positions within the morula, the polar cells being peripheral and the apolar cells being central. Since peripheral and central cells give rise to trophectoderm and inner cell mass in the blastocyst, we therefore suggest that the foundation of the trophectoderm and inner cell mass lineages may occur by a process of differential inheritance. This conclusion supports the recently proposed polarization hypothesis, which is discussed.