The aim of the present study is to determine the anti-proliferative activity of 6-o-palmitoyl-l-ascorbic acid (Asc6Palm) that is a lipophilic derivative of l-ascorbic acid (Asc), on human tongue squamous carcinoma HSC-4 cells by combined use of hyperthermia in comparison to Asc. Asc6Palm or Asc were administered to HSC-4 cells for 1 h, to which hyperthermia at 42 A degrees C was applied for initial 15 min. After further 1-72 h incubation at 37 A degrees C, cell proliferation was determined with Crystal Violet staining. Ascorbyl radical (AscR) in HSC-4 cell suspension was measured by electron spin resonance (ESR), and cell morphology was observed with scanning electron microscopy (SEM). At 37 A degrees C, 4 mM Asc or 0.35 mM Asc6Palm were enough to suppress proliferation of HSC-4 cells. By combined use of hyperthermia at 42 A degrees C, cell proliferation was decreased when compared to 37 A degrees C. After Asc of 4 mM was incubated with HSC-4 cell suspensions at 37 A degrees C or 42 A degrees C for 0-180 min, the signal intensity of ascorbyl radical (AscR) by ESR was not different regardless of the presence or absence of cells at 37 A degrees C, whereas AscR signal was enlarged in the presence of HSC-4 cells at 42 A degrees C. It was suggested that oxidation of Asc occurred rapidly in HSC-4 cells by hyperthermia, and thereby enhanced the anti-proliferative activity. By SEM observation, the surface of HSC-4 cells treated with Asc6Palm revealed distinct morphological changes. Thus, the combined regimen of Asc6Palm and hyperthermia is expected to exert a marked antitumor activity.