The region of beta-spectrin that is responsible for interactions with ankyrin was shown to comprise an ankyrin-sensitive lipid-binding site. Structural studies indicate that it exhibits a mixed 3(10)/alpha helical conformation and is highly amphipathic. These features together with the distinctively conserved sequence of the lipid-binding site motivated us to explore the mechanism of its interactions with biological membranes. A series of singly and doubly spin-labeled erythroid beta-spectrin-derived peptides was constructed, and the spin-label mobility and spin-spin distances were analyzed via electron paramagnetic resonance spectroscopy and two different calculation methods. The results indicate that in beta-spectrin, the lipid-binding domain, which is part of the 14(th) segment, has the topology of typical triple-helical spectrin repeat. However, it undergoes significant changes when interacting with phospholipids or detergents. A mechanism for these interactions is proposed in this paper.