The influence of headgroup chirality on the mixing behavior of a series of phosphatidylglycerol (PG) mimics has been investigated by use of the nearest-neighbor recognition method [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Sec. 1995, 117, 7838]. For this purpose, a series of disulfide-based phospholipid dimers 1-10 were synthesized from 1,2-dimyristoyl-sn-glycero-3-phosphate and 1,2-stearoyl-sn-glycero-3-phosphate, having the R-configuration at the sn-2 carbon, and either a R-or an S-configuration within the headgroup. Results of nearest-neighbor analyses indicate that chiral interactions between the headgroup and the glycerol backbone of neighboring PG mimics have a larger influence on lipid mixing than a chain length difference of four methylenes in analogous lipids, which are devoid of chirality and a hydroxymethylene moiety in the headgroup.