Cell surface and calf serum proteins were released in vitro from cultured virus-transformed, chemically transformed and normal rat embryo fibroblasts in 0.2 M urea containing serum-free culture medium. Fibronectin (LETS protein) was found in considerably higher amount in medium from normal rat embryo fibroblasts than in that from transformed cells. Major calf serum protein released from cultured normal and transformed rat cells corresponded by its electrophoretic mobility to calf serum albumin. No substantial differences in electrophoretic patterns of calf serum proteins released from normal and transformed cells into the medium were found. Time course of release of serum proteins from cell surface was measured quantitatively with the aid of radioiodinated Staphylococcus aureus protein A radioimmune assay. More than 50% of cell bound calf serum proteins remained on cell surface after 24 hours of incubation in serum-free medium.