Co-culture of pre-implantation embryos with cells of the reproductive tract requires a medium that is beneficial to both embryos and cells. However, many studies in this area utilize media originally formulated for specific cell lines. In the present study, a complex serum-free medium (CSM) was formulated on the basis of the ionic compositions of existing embryo culture media and mouse oviductal fluid as well as the concentrations of growth factors that appear to benefit mouse embryo development. The study began by investigating the effect of altering the concentrations of K+ ions (0-40 mM) and sulfate ions (0-10 mM) in embryo culture media on the development of 2-cell mouse embryos. Mouse embryos showed improved cell numbers at the blastocyst stage when cultured in 10 mM K+ compared with Whittingham's T6 medium. Embryos were also cultured in T6 supplemented with bovine serum albumin (BSA) containing various concentrations of insulin, insulin-like growth factors I and II, fibroblast growth factor, and epidermal growth factor. Insulin concentrations of 100 ng mL-1 significantly (P less than 0.05) improved the cell numbers of 2-cell embryos cultured to the morulae and blastocyst stages compared with those cultured in T6 + BSA alone. CSM was formulated on the basis of the results of these experiments and was found to support both improved development of 2-cell mouse embryos and the culture of mouse fibroblast and mouse oviduct cells.