The (beta/alpha)(8)-barrel is one of the most common folds functioning as enzymes. The emergence of two (beta/alpha)(8)-barrel enzymes involved in histidine biosynthesis, each of which has a twofold symmetric structure, has been proposed to be a consequence of tandem duplication and fusion of a (beta/alpha)(4)-half-barrel. However, little evidence has been found for the existence of an ancestral half-barrel in the evolution of other (beta/alpha)(8)-barrel proteins. In order to detect remnants of an ancestral half-barrel in the (beta/alpha)(8)-barrel structure of Escherichia coli N-(5'-phosphoribosyl)anthranilate isomerase, we engineered three potential half-barrel units, (beta/alpha)(1-4), (beta/alpha)(3-6), and (beta/alpha)(5-8). Among these three arrangements, only (beta/alpha)(3-6) is stable; it exists in equilibrium between monomeric and dimeric forms. Thus, the central segment of N-(5'-phosphoribosyl)anthranilate isomerase from E. coli can serve as a half-barrel precursor. A tandem duplication of (beta/alpha)(3-6) yielded predominantly monomeric structures that were quite stable. This result exemplified that the structural characteristics of noncovalently assembled half-barrels could be improved by duplication and fusion. Moreover, our results may provide information regarding the local structural units that encompass interactions important for the early folding events of this ubiquitous protein conformation.