Cell membranes are complex mixtures of lipids, proteins, and other molecules that serve as active, semipermeable barriers between cells, as well as between their internal organelles, and the surrounding medium. Their compositions and structures are tightly regulated to ensure proper function. Cholesterol is a key component in mammalian cellular membranes, where it serves to maintain membrane fluidity and permeability. Here, the interaction of alamethicin, a 20 amino acid residue peptide that creates transmembrane pores in lipid bilayer membranes in a concentration-dependent manner, with bilayer membranes composed of dimyristoyl phosphatidylcholine (DMPC) and cholesterol (Chol) was studied. Small-angle neutron scattering (SANS) data demonstrate that a low concentration of alamethicin (peptide-to-lipid ratio of 1/200) disrupts a lateral inhomogeneity seen in peptide-free DMPC:Chol vesicles, which analysis of the SANS data indicates are Chol-rich and Chol-poor phases having different thicknesses. Alamethicin disrupts this structure, producing laterally homogeneous bilayers that are thinner than either phase of the peptide-free bilayers, and possess a strong asymmetry in the Chol content of the inner and outer bilayer leaflets. The results suggest that a secondary membrane disruption mechanism exists in parallel with the well-understood cytotoxic membrane permeabilization that results when alamethcin forms transmembrane pores. Specifically, the peptide can disrupt laterally organized lipidic structures in cell membranes, as well as significantly perturb the compositions of the inner and outer leaflets of the membrane. The existence of a secondary mechanism of action against cellular membranes for alamethicin raises the possibility that other membrane-active peptides function similarly.