Higher plants possess two distinct nuclear-encoded glucose-6-phosphate isomerase (GPI) isoenzymes, a cytosolic enzmye of the Embden-Meyerhof pathway and a chloroplast enzyme essential to storage and mobilization of carbohydrate fixed by the Calvin cycle. We have purified spinach chloroplast GPI to homogeneity, determined amino acid sequences from the active enzyme, and cloned cDNAs for chloroplast and cytosolic GPI isoenzymes from spinach. Sequence comparisons reveal three distantly related families of GPI genes that are non-uniformly distributed among contemporary eubacteria and archaebacteria, suggesting that ancient gene diversity existed for this glycolytic enzyme. Spinach chloroplast GPI is much more similar to its homologue from the cyanobacterium Synechocystis PCC6803 than it is to the enzyme from any other source, providing strong evidence that the gene for chloroplast GPI was acquired by the nucleus via endosymbiotic gene transfer from the cyanobacterial antecedants of chloroplasts. Eukaryotic nuclear genes for cytosolic GPI are more similar to eubacterial than to archaebacterial homologues, suggesting that these too were acquired by eukaryotes from eubacteria, probably during the course of the endosymbiotic origin of mitochondria. Chloroplast and cytosolic GPI provide evidence for a eubacterial origin of yet another component of the eukaryotic glycolytic pathway.