HCT-8R human colon tumor cells heated for 60 minutes at 37-44.5 degrees C showed an increased sensitivity to lysis by rabbit anti-HCT-8R antibodies and complement (Ab-C) at the higher temperatures. As compared to the 51Cr release assay, the colony formation (CF) assay was a more sensitive measure of hyperthermia-induced cell damage and sensitization to Ab-C lysis. Also the CF assay was more efficient than the 51Cr assay in detecting effects of low-temperature heating (41.5 degrees C) on Ab-C cytotoxicity. The direct toxicity of heating alone was minimal. Because heating did not influence the pH of the medium, the pH of our assay system did not appear to be factor in the enhanced sensitivity of heated cells to Ab-C cytotoxicity. Significantly smaller amounts of antibodies were capable of lysing heated cells as compared to unheated cells, but heated cells did not bind more antibodies than did unheated cells. This suggests that the hyperthermia effect on the cell occurs at some stage of the lytic event after antibody binding. Hyperthermia treatment at 43.5 degrees C enhanced cytotoxicity by antibodies monospecific to carcinoembryonic antigen, which is expressed on the surface of these cells. Thus hyperthermia may be an effective tool in augmenting specific immune reactions against tumor-associated cell membrane antigens.