The earliest manifestation of the toxicity of ozone in an in vitro system utilizing human erythrocytes had been previously suggested to be due to the breakdown of unsaturated fatty acids in the cell membrane. To confirm that destruction of unsaturated fatty acids does occur in biological membranes exposed to ozone, red cell fatty acids were extracted, methylated, and quantitated by gas-liquid chromatography. After ozone exposure there was a relative decrease in unsaturated fatty acids as compared to saturated fatty acids, and the more unsaturated the fatty acid, the greater the loss. These findings are interpreted as additional evidence supporting the concept that the peroxidation or ozonization of unsaturated fatty acids in biological membranes is a primary mechanism of the deleterious effects of ozone.