Deuterium nuclear magnetic resonance spectra (at 55.3 MHz) have been obtained of 19F-2H double-labeled phospholipids in pure lipid bilayers, and of 2H-labeled lipid in a 19F-labeled bilayer, as a function of concentration, to assess the perturbing influence of 19F sites in lipid hydrocarbon chains. Order parameters of 2H-labeled sites adjacent to C-8 myristic fluorine probes in pure lipid bilayers, and 19F spin label order parameters themselves, are about 30% lower than those deduced from the use of nonperturbing 2H probes. The effect is intramolecular rather than intermolecular and presumably represents increased gauche states due to the increased size of the 19F label. This effect is consistent with the view that difluoromethylene fatty acyl chains function in a manner approximating that of unsaturated fatty acyl chains. The differences disappear in the presence of cholesterol at very high order parameters (Smol approximately 0.8 to 0.9). These results represent the first attempt at elucidating the perturbing effects of a high sensitivity probe (19F) and indicate that caution must be used when using spectroscopic probes to deduce the absolute magnitude of hydrocarbon chain order parameters.