The human colon, intratunmral subpopulations HCT 116 and IICI 116a were established in chemically defined medium supplemented with transfcrrin, insulin, epidermal growth factor (EGF), triiodothyronine, hydrocortisone, and sodium selenite. The responsiveness of the adapted cell lines to these growth factors was compared in anchorage-dependent and -independent assays. HCT 116 cells maintained in serum-free con ditions were further adapted to growth factor deprivation, and the effects of these polypeptides were determined in anchorage-independent assays. In monolayer, IK T 116 cells adapted to grow in serum-free medium responded to transferrin but not to EGF or insulin. Similarly adapted HCT I I6a cells were, however, insensitive to transferrin addition but manifested a 300 and 500% increase in growth rates with EGF and insulin, respectively. Optimal growth of HCT 116 cells was seen in the presence of insulin and transferrin, while maximum proliferation of HCT I I6a cells depended on combined insulin, transferrin, and EGF. In soft agarose, both HCT 116 and HCT 116a subpopulations showed a stringent requirement for transferrin. No combination of growth factors without transfemn supported colony formation. These data suggest that (a) these colon tumor subpopulations may be subject to separate growth controls, and (b) there may be an important role for transferrin in anchorage-independent growth and possibly in the maintenance of malignant characteristics.