A serumless medium suitable for prolonged growth and repeated sub-culture of Walker carcinosarcoma 256, sarcoma 180, KB and human heart cells in vitro was described. In addition to amino acids, vitamins, minerals and glucose, it contained insulin, salmine sul-fate, pyruvate, mucate, methyl oleate, folinic acid and lactalysate. The latter materials with exception of mucate were shown to be required for maximal growth rates, comparable to those on media containing dialyzed serum. Suspension cultures of Walker carcinosarcoma 256 also were grown with continuous passage in the serumless medium.