The evolutionary relationships of proteobacteria, which comprise the largest and phenotypically most diverse division among prokaryotes, are examined based on the analyses of available molecular sequence data. Sequence alignments of different proteins have led to the identification of numerous conserved inserts and deletions (referred to as signature sequences), which either are unique characteristics of various proteobacterial species or are shared by only members from certain subdivisions of proteobacteria. These signature sequences provide molecular means to define the proteobacterial phyla and their various subdivisions and to understand their evolutionary relationships to the other groups of eubacteria as well as the eukaryotes. Based on signature sequences that are present in different proteins it is now possible to infer that the various eubacterial phyla evolved from a common ancestor in the following order: low-G+C Gram-positive-->high-G+C Gram-positive-->Deinococcus-Thermus (green nonsulfur bacteria)-->cyanobacteria-->Spirochetes-->Chlamydia-Cytophaga-Aquifex -green sulfur bacteria-->Proteobacteria-1 (epsilon and delta)-->Proteobacteria-2 (alpha)-->Proteobacteria-3 (beta)-->Proteobacteria-4 (gamma). An unexpected but important aspect of the relationship deduced here is that the main eubacterial phyla are related to each other linearly rather than in a tree-like manner, suggesting that the major evolutionary changes within Bacteria have taken place in a directional manner. The identified signatures permit placement of prokaryotes into different groups/divisions and could be used for determinative purposes. These signatures generally support the origin of mitochondria from an alpha-proteobacterium and provide evidence that the nuclear cytosolic homologs of many genes are also derived from proteobacteria.