Study of the most conserved region in many @/a-barrels, the phosphate-binding site, revealed a sequence motif in a few @/a-barrels with known tertiary structure, namely glycolate oxidase (COX), cytochrome b2 (Cyb2), tryptophan synthase a subunit (TrpA), and the indoleglycerolphosphate synthase (TrpC). Database searches identified this motif in numerous other enzyme families: (1) IMP dehydrogenase (IMPDH) and GMP reductase (GuaC); (2) phosphoribosylformimino-5-aminoimidazol carboxamide ribotide isomerase (HisA) and the cyclaseproducing D-erythro-imidazole-glycerolphosphate (HisF) of the histidine biosynthetic pathway; (3) dihydroorotate dehydrogenase (PyrD); (4) glutamate synthase (GltB); ( 5 ) ThiE and ThiG involved in the biosynthesis of thiamine as well as related proteins; (6) an uncharacterized open reading frame from Erwinia herbicola; and (7) a glycerol uptake operon antiterminator regulatory protein (GlpP). Secondary structure predictions of the different families mentioned above revealed an alternating order of @-strands and a-helices in agreement with a @/a-barrel-like topology. The putative phosphate-binding site is always found near the C-terminus of the enzymes, which are all at least about 200 amino acids long. This is compatible with its assumed location between strand 7 and helix 8. The identification of a significant motif in functionally diverse enzymes suggests a divergent evolution of at least a considerable fraction of @/a-barrels. In addition to the known accumulation of @/a-barrels in the tryptophan biosynthetic pathway, we observe clusters of these enzymes in histidille biosynthesis, purine metabolism, and apparently also in thiamine biosynthesis. The substrates are mostly heterocyclic compounds. Although the marginal sequence similarities do not allow a reconstruction of the barrel spreading, they support the idea of pathway evolution by gene duplication.