The adhesion and growth of mouse embryonic stem cells line D3 were tested in polystyrene culture dishes coated with the multilayer assemblies containing various cell-adhesive proteins such as gelatin, collagen IV, and laminin or polycations polylysine and poly(ethyleneimine). The cells were seeded in complete serum-containing media or in serum-free media and in the presence (non-differentiated) and/or absence (differentiated) of leukemia inhibitory factor. The cells grew best on surfaces coated with gelatin and collagen IV assemblies. There were no significant differences in the growth of the non-differentiated and differentiated cells in complete serum-containing media. When seeded in serum-free media, non-differentiated cells grew better than the differentiated ones. The layer-by-layer deposition appears to be a practicable technique by which scaffolds for tissue engineering can be coated with biomolecular assemblies tailored to specific cells and applications.