Low-angle and wide-angle X-ray diffraction was used to examine the phase behavior, as a function of temperature, of fully hydrated multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) labeled with a difluoromethylene group in the sn-2 acyl chain at position 4, 8, or 12. At temperatures near 5 degrees C, DMPC and 2-[12,12-F-2]DMPC are both in the L(beta’) (gel) phase with a D-spacing of 59.5 +/- 0.5 Angstrom, 2-[8,8-F-2]DMPC is in the P-beta’ (rippled) phase with a D-spacing of 65.0 +/- 0.5 Angstrom, and 2-[4,4-F-2]DMPC is in the L(c) (subgel) phase with a D-spacing of 56.5 +/- 0.5 Angstrom. Thus the greatest bilayer perturbation is caused by labeling with fluorine closest to the head group region. It was found that 2-[4,4-F-2]DMPC converted from the L(alpha) (fluid) phase to the subgel phase at temperatures as high as 19.3 degrees C when incubated for 16 h at 19.3 degrees C. A hypothesis is presented that high concentrations of phospholipid in water (25%, w/w) facilitate the rapid formation of the subgel phase in 2-[4,4-F-2]DMPC. This hypothesis is consistent with the nucleation and growth mechanism of subgel formation as was shown for subgel formation in dipalmitoylphosphatidylcholine (DPPC).(1) When the subgel phase of 2-[4,4-F-2]DMPC was melted over the course of several days, phase coexistence of the subgel and fluid phases was observed at 23.1 degrees C without observation of intermediary gel or rippled phases.