OstA (Imp) homologues have been shown to play a role in outer membrane biogenesis. Bioinformatic analyses of these proteins in organisms with fully sequenced genomes reveal that these proteins occur only in bacteria with two membranes. Two OstA types were identified, large OstAs (L; 812 ± 94 residues) and small OstAs (S; 181 ± 25 residues). S possesses only the OstA domain while L has this domain plus a larger nonhomologous OstA-C domain. Bacteria lacking both S and L proteins were primarily restricted to reduced genome size pathogens and symbionts. Several of these bacteria appear to also have incomplete sets of genes required for the biosynthesis of typical Gram-negative bacterial lipopolysaccharide (LPS). Phylogenetic analyses of both S and L homologues showed that they generally follow the phylogenies of the 16S rRNAs from the same organisms with few exceptions. They may comprise two orthologous sets of proteins that together facilitate a single unified function. While most organisms possess a single L and a single S, those lacking S but possessing L are more numerous than those lacking L but possessing S. Based on our findings and those of others, we suggest that (1) the L and S proteins are nonessential for outer membrane assembly, (2) they normally act together in macromolecular insertion, (3) they are important for proper LPS assembly in the outer leaflet of the outer membrane, (4) they function specifically to export LPS to the outer leaflet, and (5) L provides a primary function while S provides an important auxiliary function.