Abstract: The electron spin resonance (ESR) spectra of a series of fatty acids bearing the paramagnetic pyrrolidin-N-oxyl ring have been studied in oriented bilayers of dimyristoylphosphatidylcholine:cholesterol (70:30 mol/mol) at pH 9.0. Nitroxyl fatty acids with the alkylcarboxy and alkyl substituents located respectively at the 2,2-, 3,2-, 2,4-, or 2,5-positions of the pyrrolidine ring were used. These disubstitutions correspond to different modes of incorporation of the nitroxyl ring into the chain. The order parameter of the nitroxyl z axis (principal hyperfine tensor element A(zz) = 32 G), S(NO), was determined from the angular dependence of ESR spectra recorded at temperatures that were demonstrated to be in the motional narrowing regime. Combination of S(NO) with the segmental order parameters of the chain axis, S(mol), allowed determination of the orientation, theta, of the nitroxyl z axis with respect to the chain diffusion axis in fluid lipid bilayers. The values of S(mol) were obtained from the ESR spectra of the appropriate positional isomers of n-(4,4-dimethyloxazolidin-N-oxyl)stearic acid, for which theta = 0. Values of theta = 0, 90, 40 +/- 5, and 65 +/- 5-degrees were obtained for the 2,2-, 2,4-, 2,5-, and 3,2-disubstituted pyrrolidine fatty acids, respectively. Comparison with molecular modeling suggests that the rotational diffusion axis lies parallel to the fatty acid trans-chain axis for all molecular conformations except that of the 2,5-disubstituted fatty acid. This spectroscopic characterization of the pyrrolidine-based fatty acids is necessary for their use as lipid spin label probes in biological membranes.