Several fluorinated fatty acids of the general structure CH3(CH2)13--mCF2(CH2)m--2COOH are incorporated biosynthetically as unsaturated fatty acid analogues into the phospholipids of Escherichia coli. Under optimum conditions an unsaturated fatty acid autotroph, K1060B5, can be grown so that 50% of the total phospholipid fatty acids are 8,8-difluoromyristate. Conditions are found for which more than 20% of the fatty acids are fluorinated before a decrease in growth rate is observed. We have used 19F nuclear magnetic resonance to examine membranes isolated from E. coli grown under the latter conditions. A comparison is made with spectra of aqueous dispersions of extracted E. coli phospholipids and model multilayer phospholipid membranes. An explanation of the 19F resonance line shape in these membrane systems and the relationship to a molecular order parameter is given. It is apparent that 19F nuclear magnetic resonance is more sensitive to the degree of ordering or fluidity of phospholipids than spin labels or fluorescent probes. For instance, a dramatic effect of membrane protein on lipid fluidity can be seen. Finally, this method can be used to measure the proportion of frozen and fluid lipid in biological membranes at temperatures within the span of the gel-to-lipid phase transition.