Despite the frequency of both oxidant drug-induced and spontaneous Heinz body formation in cats, the cellular and biochemical mechanisms by which Heinz bodies result in red blood cell (RBC) destruction and hemolytic anemia in this species remain unknown. Feline spleens are non-sinusoidal and inefficient at removing Heinz body-containing RBC from the circulation; therefore, alternative mechanisms must be involved in accelerated RBC destruction. Propylene glycol (PG) ingestion causes dose-dependent Heinz body formation and decreased RBC survival in cats. We investigated several aspects of Heinz body-containing RBC from three cats ingesting diets that provided 8.0 g PG/kg body weight for up to 3 weeks, in order to characterize cellular lesions that are associated with the presence of Heinz bodies and that might contribute to chronic, accelerated RBC destruction, as well as to gain insight into the mechanism by which PG induces Heinz body formation. Erythrocytes with PG-induced Heinz bodies had decreased levels of reduced glutathione and adenosine triphosphate and reduced deformability. There was no change in hemoglobin isoelectric focusing or membrane lipid peroxidation. Electrophoretic patterns of Heinz body-containing RBC membranes were significantly altered, and membrane surface charge distribution was disturbed. Progressively protruding Heinz bodies suggested that extrusion of Heinz bodies may be a means of cell healing and/or destruction in the absence of splenic pitting. When compared to results obtained using RBC from cats treated with the oxidant drug phenylhydrazine, significant differences were noted in packed cell volume, turbidity index, membrane heme, and morphologic appearance of Heinz bodies. Our results indicate that multiple cellular abnormalities develop in RBC with PG-induced Heinz bodies that do not cause acute hemolysis but that may shorten RBC survival. Propylene glycol-induced Heinz bodies provide an ideal model for studying the chronic effects of Heinz bodies on RBC structure and function and may be useful in understanding the mechanisms of formation and the consequences of endogenous Heinz bodies in cats.