The effect of heat on the density of cell surface histocompatibility antigens was examined. Antigen density and distribution were determined by radioimmunoassay and flow cytometry after the binding of radioiodinated or fluoresceinated monoclonal antibody (anti-H-2Kk and anti-H-2Kb) to murine lymphoma cells in suspension cultures. Antibody binding was unaffected by temperatures between 37 degrees and 41 degrees following a 30-min heat exposure. At 42 degrees, some inhibition of binding was measurable. However, at 43 degrees, antibody binding was reduced by 30 to 50%, and a further 15 to 20% reduction was observed at 45 degrees. Flow cytometry showed that all cells were equally affected. There was no indication of the selection of a specific cell population. The temperature-dependent decrease in antibody binding was due to a decrease in receptor number and not to changes in the affinity. Measurement of the diffusion coefficient of the lipid probe N,N-dioctadecyl indocarbocyanine iodide showed that heat did not affect significantly the fluidity of the membrane lipids. Hyperthermic temperatures, therefore, have a direct effect on these membrane proteins.