Inactivating mutations and/or deletions of PHEX ( Phosphate-regulating gene with Homologies to Endopeptidase on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans. In the present study, three Drosophila PHEX homologues (dPHEX-1, -2, -3) were isolated by the screening of a Drosophila cDNA library and expressed sequence tag (EST) database. The structural region involving motif II: (456)WMXXXTKXXAXXK(468) (numbered according to human PHEX), motif VI: (602)WW(603), and motif VIII: (746)CXLW(749) was conserved in the dPHEX family. Zinc-coordinating motifs (HEFTH and GENIADNGG) were also conserved in the dPHEX family. All three dPHEX genes were expressed during all stages of Drosophila development. The expression of dPHEX-1 was suppressed by dietary phosphate deprivation, but the expression of dPHEX-2 and that of dPHEX-3 were not affected. In-situ hybridization showed a ubiquitous distribution of dPHEX-1 and dPHEX-2, while dPHEX-3 was highly expressed in the larval brain. In an analysis of subcellular localization, dPHEX-1 was localized to intracellular organelles and dPHEX-3 was localized predominately in the plasma membrane of Drosophila embryonic S2 cells. Homozygosity of a dPHEX-1 mutation, a transposon insertion in the dPHEX-1 promoter region, was completely lethal at an early stage of embryonic development. The present study indicates that three homologues are likely involved in the phosphate homeostasis of Drosophila.