Transplantable mouse teratomas are known to contain multipotential teratocarcinoma stem cells analogous to early embryo cells and capable of giving rise to a wide variety of specialized cell types in vivo when they attach to a substratum; if they are grown instead in suspension in the body cavity, they form multicellular embryoid bodies which proliferate with little or no cell specialization. Thus, changes initiated at the cell surface may play some role in promoting early cell differentiation. In order to establish an in vitro system for experimental investigation of this hypothesis, embryoid body cells were explanted under conditions of cell attachment versus suspension and maintained in primary culture. Because cell differentiation in previous reports was relatively limited in vitro, the two cellular populations were first compared for genesis of a quantifiable macromolecular phenotype, acetylcholinesterase (AChE) activity, which characterizes several of the cell types most commonly formed in the attached tumors in vivo. The attached cells produced markedly increased levels of AChE activity within a few weeks, while cells in suspension retained basal levels. AChE was histochemically visualized and was found to occur chiefly in cells undergoing myogenesis, especially during myotube formation. Aberrant muscle fibers formed and became predominant in the cultures. When embryoid bodies were first fractionated by increasing size, which reflects their progressive differentiation, the smallest ones, with relatively more multipotential cells and no apparent muscle cells, also showed AChE increase in attached cultures. The results are consistent with the view that attachment of the cell surface to a substratum may play a critical role in initiating some cellular developmental commitments, as well as in sustaining differentiation of cells whose specialization has already been determined. Further experimental modifications of this primary culture system of teratocarcinoma cells should be useful in analyzing cell-substratum relations and cell-surface changes in early mammalian development.