Procaine, a membrane-specific agent, was found to protect the response of oxygenated mammalian cells toward X irradiation in vitro. In addition, the drug also potentiates hyperthermic killing of cells. In an effort to gain further insights into the mechanisms underlying the action of procaine as a modifier of cell injury in X-irradiated and heated cells, the effects of procaine on cellular morphology, membrane fluidity, and cellular electrokinetic properties were studied. It was found that procaine can reversibly influence the cellular morphology. Using scanning electron microscopy, the authors were able to demonstrate the presence of numerous smooth blebs on the cell surface of either normal or SV40-transformed 3T3 mouse fibroblasts treated with procaine. When 1,6-diphenyl-1,3,5-hexatriene was used as a membrane probe, the overall fluidity of cell membranes from 3T3 cells grown as monolayers or L5178Y lymphoma cells grown in suspensions was not significantly changed by procaine; however, when perylene was used as a probe, the overall fluidity of cell membranes of procaine-treated cells was found to increase significantly. Lastly, procaine was found to alter the electrophoretic mobility of cells. These results indicate that procaine may exert its modification of radiation- and heat-induced effects via a still elusive membrane-mediated mechanism(s). It is anticipated that membrane-active agents with different specificities may be employed as tools to probe the role of membranes in the radiation or heat response of cells.