Polymorphonuclear leukocytes from humans and mice with the Chediak-Higashi syndrome were characterized by spin label electron spin resonance spectrometry. Our results suggest that cells from afflicted mice and humans have membranes more fluid than controls. Order parameters for a spin label that probes near the membrane surface were 0.652 for normals and 0.645 for two Chediak-Higashi patients. Cells from Chediak-Higashi mice showed similar differences, as did isolated plasma membrane fractions. An increased membrane fluidity was also detected with a spin label that probes deeper in the bilayer. In vitro treatment of Chediak-Higashi mouse cells with 0.01 M ascorbate increased the order parameter to normal levels. In vitro incubation of mouse Chediak-Higashi cells with glucose oxidase increased the order parameter, similar to the effect of ascorbate. This increase was abolished when catalase was added to the incubation medium. In vitro incubation with dibutyryl cyclic guanosine monophosphate (1 μM to 0.1 mM) did not normalize order parameters. These results indicate that fluidity of Chediak-Higashi cell membranes was affected by treatments expected to alter the oxidation: reduction potential of the environment but was not affected by treatments expected to alter the ratio of intracellular cyclic nucleotides. The latter treatment would affect microtubule assembly. Therefore, it appears that the membrane fluidity abnormalities as demonstrated by electron spin resonance and the earlier demonstrated microtubule dysfunctions characteristic of Chediak-Higashi cells are coexisting defects and are probably not directly related.