Forelimb buds of rat embryos at day 13 of gestation were cut and cultured in Dulbecco's modified Eagle medium. In the absence of serum, the epidermis did not cover the wound surface. In the presence of fetal calf serum or ultroser G, a serum substitute, epidermal wound closure appeared in a concentration-dependent manner within 1 day of culture. Cycloheximide, an inhibitor of protein synthesis, reversibly inhibited wound healing, demonstrating that protein synthesis was required for epidermal wound closure. Dialysis and fractionation of ultroser G indicated that high molecular weight factors (greater than 25,000 d) were required for epidermal wound closure.