The study was designed to show whether the increase in unsaturated fatty acids in Tetrahymena pyriformis membrane phospholipids with decreasing growth temperature is due to the sharp rise in the concentration of dissolved O2 found at lower temperatures. Increasing cell density in cultures growing at a constant temperature was found to have a more drastic effect upon the O2 level than shifting a culture from one temperature extreme to the other. However, the pattern of phospholipid fatty acid unsaturation did not vary over a 30-fold gradual decrease in O2 concentration measured in cultures during logarithmic growth at 39.5 degrees C, and unsaturation in 15 degrees C cells sampled over a 5-fold decrease in O2 concentration was also unchanged. Cells grown at 39.5 degrees C under a constant relatively high O2 concentration (5--6 mg O2/l) or low O2 concentration (0.3 mg O2/l) had nearly identical distributions of membrane fatty acids. Only under almost completely anaerobic conditions was it possible to measure via radioactive labeling experiments and inhibition of fatty acid desaturation. Thus the increased membrane fatty acid unsaturation induced by decreasing growth temperature is caused by some factor other than the rising levels of dissolved O2.