Endonexin (annexin IV) is a member of the annexin family of homologous proteins that share the ability to bind to pure lipid membranes and to aggregate vesicles in a Ca(2+)-dependent fashion. Endonexin appears to preferentially interact with certain types of lipids such as phosphatidylglycerol (PG) in PG/phosphatidylcholine (PC) mixed lipid membranes. Such preferential binding should result in localization of PG lipids to membrane regions where endonexin is bound. This was tested using a PG derivative containing the fluorophore pyrene, which exhibits fluorescence sensitive to molecular collision frequency. Motional restriction of pyrene-PG upon endonexin-membrane binding was evident from decreased ratios of excimer-to-monomer (E/M) pyrene fluorescence with endonexin binding to 97% PG/3% pyrene-PG vesicles. A maximum decrease of 30% suggests a 30% decrease in the average diffusion constant of pyrene-PG molecules or a 53% decrease assuming that only outer-monolayer lipid molecules interact with endonexin. In vesicles containing 5% and 10% pyrene-PG in PC, segregation of lipids was evident from observed increases in E/M of 14.2 +/- 1.8% and 6.8 +/- 0.1%, respectively, in the presence of endonexin and either 10 mM (5% pyrene-PG) or 2 mM (10% pyrene-PG) free Ca2+. At higher concentrations of Ca2+ (> 10 mM for 5% pyrene-PG and > 2 mM for 10% pyrene-PG), smaller endonexin-dependent increases in E/M are observed as endonexin molecules at high surface densities compete for the limited pool of pyrene-PG. The nature of these interactions of endonexin with mixed lipid systems has implications for the way annexins may modulate membrane structure in cells.