We examined structural and functional properties of the recently duplicated cytosolic isozymes of phosphoglucose isomerase (PGI) (EC 5.3.1.9) of the wild plant Clarkia xantiana and related species. A new purification protocol yielded PGIs with high specific activities. The duplicated PGI isozymes showed similar substrate affinities (apparent Km) in both catalytic directions. A newly devised competitive enzyme-linked immunosorbent assay (ELISA), using polyclonal antibodies, distinguished sodium dodecyl sulfate (SDS)-denatured PGIs coded by the duplicated loci but did not discriminate between allelic products of one of the loci. In vitro dissociation and reassociation experiments revealed that the duplicated subunits differed in their efficiency of reassociation. The difference was closely correlated with differences between the isozymes in their in vivo accumulation. In contrast, allelic subunits in species with or without the duplication were able to reassociate with similar efficiency. The duplicated PGI isozymes have diverged more in structural properties than kinetic ones. The total evidence suggests that mechanisms have evolved which reduce the potential consequences of the duplication.