In the gram-negative model organism Escherichia coli, the effector molecule of the stringent response, (p)ppGpp, is synthesized by two different enzymes, RelA and SpoT, whereas in the gram-positive model organism Bacillus subtilis only one enzyme named Rel is responsible for this activity. Rel and SpoT also possess (p)ppGpp hydrolase activity. BLAST searches were used to identify orthologous genes in databases. The construction and bootstrapping of phylogenetic trees allowed classification of these orthologs. Four groups could be distinguished: With the exception of Neisseria and Bordetella (beta subdivision), the RelA and SpoT groups are exclusively found in the gamma subdivision of proteobacteria. Two Rel groups representing the actinobacterial and the Bacillus/Clostridium group were also identified. The SpoT proteins are related to the gram positive Rel proteins. RelA proteins carry substitutions in the HD domain (Aravind and Koonin, 1998, TIBS 23: 469-472) responsible for ppGpp degradation. A theory for the evolution of the specialized, paralogous relA and spoT genes is presented: After gene duplication of an ancestral rellike gene, the spoT and relA genes evolved from the duplicated genes. The distribution pattern of the paralogous RelA and SpoT proteins supports a new model of linear bacterial evolution (Gupta, 2000, FEMS Microbiol. Rev. 24: 367-402). This model postulates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage. However, two paralogous, closely related genes of Porphyromonas gingivalis (Cytophaga-Flavobacterium-Bacteroides phylum) encoding proteins with functions probably identical to the RelA and SpoT proteins do not fit in this model. Completely sequenced genomes of several obligately parasitic organisms (Treponema pallidum, Chlamydia species, Rickettsia prowazekii) and the obligate aphid symbiont Buchnera sp. APS as well as archaea do not contain rel-like genes but they are present in the Arabidopsis genome. In crosslinking experiments using different analogs of ppGpp as crosslinking reagents and RNA polymerase preparations of Escherichia coli, binding of ppGpp to distinct regions at the C-terminus of the beta subunit (the RpoB gene product) and/or at the N-terminus of the beta subunit (the RpoC gene product) was observed previously. RpoB and RpoC sequences of the species which do not possess a rel like gene do not exhibit specific insertions or deletions in the ppGpp binding regions.