The effect of riboflavin deficiency on the fluidity and function of the red blood cell (RBC) membrane and on the activity of some enzymes involved in antioxidant defense mechanisms was studied. Growing male rats were fed an experimental (riboflavin-deficient) or a control (riboflavin-supplemented) diet. Following 7 wk of feeding, RBC from riboflavin-deficient rats contained higher levels of peroxidation products, most likely due to decreased glutathione reductase activity. An elevation in glutathione peroxidase activity was also observed whereas the activity of catalase and superoxide dismutase was not affected. Membrane fluidity was studied by fluorescence polarization, using 1,6-diphenyl-1,3,5 hexatriene (DPH) as a probe. The fluidity of RBC membranes isolated from riboflavin-deficient rats was significantly lower than that of the controls. This decreased fluidity was accompanied by an increase in the activity of the membrane-bound enzyme acetylcholinesterase. This study demonstrated that a decrease in cells' ability to cope with peroxidative damage as a result of riboflavin deficiency may lead to changes in the fluidity and function of membranes.