Specifically deuterated palmitic acid was incorporated into the membrane phospholipids of the L51 strain of Escherichia coli. The cytoplasmic and outer membranes were separated by using standard techniques and studied by deuterium nuclear magnetic resonance between 0 and 40 degrees C. Distinctive liquid-crystalline and gel spectra were observed to coexist over a wide temperature range. The relative intensities of these spectra provided a direct measure of the fraction of the deuterium-labeled phospholipids in the fluid state as a function of temperature. Above 37 degrees C, the amount of immobilized or gel-phase phospholipid is estimated to be less than 3% of the total phospholipid. The gel to liquid-crystalline transition region for the outer membrane was shifted upwards by approximately 7 degrees C relative to that of the cytoplasmic membrane, in agreement with previous studies [Davis, J. H., Nichol, C. P., Weeks, G., & Bloom, M. (1979) Biochemistry 18, 2103]. The orientational order in the fluid phase of both membranes decreased gradually with increasing temperature and was greater in the outer membrane than in the cytoplasmic membrane. The orientational order of the gel-phase component was the same for both membranes, within an experimental uncertainty of 10%, and was independent of temperature from 0 to 30 degrees C for the outer membrane and from 10 to 30 degrees C for the cytoplasmic membrane.