Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908 Received June 14, 1991 abstract: Annexin self-association was studied with 90 light scattering and resonance energy transfer between fluorescein (donor) and eosin (acceptor) labeled proteins. Synexin (annexin VII), p32 (annexin IV), and p67 (annexin VI) self-associated in a Ca2+-dependent manner in solution. However, thisactivity was quite labile and, especially for p32 and p67, was not consistently observed. When bound to chromaffin granule membranes, the three proteins consistently self-associated and did so at Ca2+ levels (pCa 5.0-4.5) approximately 10-fold lower than required when in solution. Phospholipid vesicles containing phosphatidylserine and phosphatidylethanolamine (1: 1 or 1: 3) were less effective at supporting annexin polymerization than were those containing phosphatidylserine and phosphatidylcholine (1: 0, 1: 1, or 1: 3). The annexins bound chromaffin granule membranes in a positively cooperative manner under conditions where annexin self-association was observed, and both phenomena were inhibited by trifluoperazine. Ca2+-dependent chromaffin granule membrane aggregation, induced by p32 or synexin, was associated with intermembrane annexin polymerization at Ca2+ levels less than pCa 4, but not at higher Ca2+ concentrations, suggesting that annexin self-association may be necessary for membrane contact at low Ca2+ levels but not at higher Ca2+ levelswhere the protein may bind two membranes as a monomer. e annexins are a newly described group of homologous proteins that bind phospholipid membranes in a Ca2+-dependent manner [for reviews see Klee (1988), and Burgoyne and Geisow (1989)]. Some members of this group are also commonly known as lipocortins (Huang et al., 1986), cal-pactins (Glenney, 1986), chromobindins (Creutz et al., 1983, 1987), calelectrins (Sudhof et al., 1984), or placental anticoagulant proteins (Tait et al., 1988). Currently, 10 distinct members of this gene family, termedannexins IX, have been identified (Pepinsky et al., 1988; Burns et al., 1989; Haupt-mann et al., 1989; Johnston et al., 1990). Comparison of their amino acid sequences reveals a common structural theme: Each protein has two regions, a variable-length amino-terminal region lacking homology with other members of the family and a core region of four or eight repeating 70 amino acid domains, which share 40-60% homology between family members.