The mixing behavior of three saturated ester phospholipids with one ether analog has been examined in the bilayer state by use of nearest-neighbor recognition methods [Vigmond, S. J.; Dewa, T.; Regen, S. L. J. Am. Chem. Sec. 1995, 117, 7838]. Specifically, the miscibility of 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and 1,2-distearoyl-sn-nglycero-3-phosphoethanolamine (DSPE) with 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE) has been investigated by analyzing mixtures of corresponding dithidipropionyl-based dimers that have been chemically equilibrated via thiolate-disulfide interchange reactions. In the fluid phase, those membranes derived from DMPE plus DHPE were found to exhibit lateral heterogeneity. In contrast, fluid membranes that were derived from DPPE plus DHPE, and also DSPE plus DHPE, were randomly arranged. Inclusion of cholesterol did not significantly alter the lateral distribution of any of these phospholipid mixtures in the fluid phase. Comparison with results obtained from previous nearest-neighbor recognition studies has revealed a synergistic effect between an ester/ether and chain length mismatch, which yields a nonrandom state. Lateral heterogeneity within bilayers derived from DMPE plus DHPE was also evident in the gel-fluid coexistence region from NNR and from high-sensitivity differential scanning calorimetry (hs-DSC) analyses; such heterogeneity, however, was eliminated by the presence of 40 mol % cholesterol and was significantly reduced in the presence of 50 mol % DPPC. Analogous gel-fluid membranes derived from DPPE plus DHPE, and DSPE plus DHPE, were found to favor a random arrangement in the gel-fluid phase. The biological significance of these findings are briefly discussed.