Membranes play a crucial role in many cellular processes, and it is therefore not surprising that many electron tomographic studies in life sciences concern membranous structures. While these tomographic studies provide many new insights into membrane connections and continuities in three dimensions, they are mostly limited to a macro-morphological level. In this paper, we demonstrate that by combining electron tomography and three-dimensional template matching we are able to investigate membrane morphology at a new level: membrane domains in three dimensions. To test this, temperature induced lipid phase separation in the biological model system of the Escherichia coli bacteria was used. We compared the inner (containing phospholipids) and outer (containing lipopolysaccharides) leaflet of the E. coli outer membrane at both 37 and -20 degrees C, and could visualize how these leaflets react differently to temperature shifts. These findings can be explained by the physico-chemical nature of the building blocks and are in line with earlier published data. This study shows that the combination of electron tomography and template matching is robust enough to visualize membrane domains that are beyond the perception of manual annotation. (C) 2009 Elsevier Inc. All rights reserved.