We have derived serum-free mouse embryo (SFME) cultures in a basal nutrient medium supplemented with insulin, transferrin, epidermal growth factor (EGF), high-density lipoprotein (HDL), and fibronectin. These cells are nontumorigenic, lack gross chromosomal aberrations, and exhibit several other unique properties, including dependence on EGF for survival and growth inhibition by serum. We have examined the concentration dependence of the growth stimulatory effects of protein supplements used in the SFME medium formulation and surveyed other supplements that might act as alternative or complementary additions to the culture medium. Insulin could be replaced by insulin-like growth factor I and EGF could be replaced by transforming growth factor alpha in the same concentration range. Transferrin could be replaced by higher concentrations of lactoferrin. Deterioration of cultures in the absence of EGF began within 8 hours of the removal of the growth factor, and could be prevented by the addition of fibroblast growth factor/heparin-binding growth factor. Attachment proteins other than fibronectin were effective on SFME cells, but limited success was obtained when substituting other lipid preparations for HDL. These data introduce a precise system for exploring the unusual characteristics of SFME cells and contribute additional information that may be useful in the extension of these approaches to other cell types and species.