Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8-and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.Halophilic and halotolerant microorganisms have adapted different methods for withstanding the high osmotic pressure exerted by their surrounding hypersaline environment. Halophilic archaea (7, 13), as well as the halophilic bacterium Salinibacter ruber (19), maintain a high intracellular salt concentration by accumulating K ϩ and Cl Ϫ ions and excluding Na ϩ ions, thus requiring intracellular proteins to be active under high-salt conditions. Many halophilic bacteria (24), the halotolerant green alga Dunaliella sp. (3), and some haloarchaea (12) exclude cytoplasmic salts and rely on organic solutes such as ectoine, glycine betaine, and glycerol to provide osmotic balance. Glycerol, in particular, is accumulated in molar quantities by Dunaliella as an organic osmotic solute. Due to leakage from healthy Dunaliella cells (1, 3, 26) and/or cellular lysis, glycerol is released into the surrounding environment, where it serves as a primary energy source for haloarchaea. Upon uptake, halophilic microorganisms assimilate glycerol into dihydroxyacetone phosphate (DHAP) by one of two catabolic routes (Fig. 1). In one route, glycerol is first phosphorylated by glycerol kinase to form sn-glycerol-3-phosphate, which is subsequently oxidized by sn-glycerol-3-phosphate dehy...