The study of cell membrane lipid and steroid composition and distribution is important for the understanding of membrane dynamics and function. Here we present efforts to chemically image phospholipid distributions on a submicron scale on freeze-fractured and frozen-hydrated liposomes and red blood cells using time-of-flight secondary ion mass spectrometry. Sample preparation by freeze fracturing of membranes is described. Fragments representative of phospholipid headgroups are found to be localized on both liposomes and red blood cells. In addition, the cholesterol molecular ion [M + H] is localized on liposome surfaces.