A cluster of genes essential for the biosynthesis of carotenoids in Erwinia herbicola has been isolated and characterized [Armstrong, G.A., Alberti, M. & Hearst, J. E. (1990) Proc. Natl. Acad. Sci. USA 87, 9975-9979]. Related gene clusters are found in other carotenoid-producing bacteria. Two of these genes, crtB and crtE, have been assigned to enzymes responsible for conversion of geranylgeranyl diphosphate (GGPP) to prephytoene diphosphate and prephytoene diphosphate to phytoene, respectively. We isolated crtE from the Er. herbicola cluster by PCR amplification and cloned the coding region into the Escherichia coli expression vector pARC306N. Es. coli JM101 was transformed with the expression plasmid, and transformants were assayed for GGPP synthase and phytoene synthase activity. Extracts from JM101/pSM145 accumulated [14C]GGPP when incubated with [14C]isopentenyl diphosphate and farnesyl diphosphate, whereas similar incubations with [3H]GGPP did not yield prephytoene diphosphate or phytoene. Thus, crtE encodes GGPP synthase.