A synthetic ionophore (1) has been prepared via condensation of 23,24-bisnor-5-cholcnic acid with both amino groups of 1,17-diamino-3,6,9,12,15-pentaoxaheptadecane and used to probe the mixing behavior of a series of unsaturated phosphocholines (PCs) in the: fluid bilayer state. Specifically, the ability of 1 to promote the transport of Na+ across membranes made from equimolar mixtures of "long" and "short" PCs has been compared with that in analogous membranes derived from single-component PCs of intermediate length. Relative ionophoric activities have been measured by use of Na-23(+) NMR spectroscopy; The results of this study provide compelling evidence that a hydrophobic mismatch of four methylene groups can lead to nonideal mixing of phosphocholines in fluid bilayers.