The hyperthermophilic archaeon Pyrococcus furiosus grows optimally at 100°C by the fermentation of peptides and carbohydrates. Growth of the organism was examined in media containing either maltose, peptides (hydrolyzed casein), or both as the carbon source(s), each with and without elemental sulfur (S 0 ). Growth rates were highest on media containing peptides and S 0 , with or without maltose. Growth did not occur on the peptide medium without S 0 . S 0 had no effect on growth rates in the maltose medium in the absence of peptides. Phenylacetate production rates (from phenylalanine fermentation) from cells grown in the peptide medium containing S 0 with or without maltose were the same, suggesting that S 0 is required for peptide utilization. The activities of 14 of 21 enzymes involved in or related to the fermentation pathways of P. furiosus were shown to be regulated under the five different growth conditions studied. The presence of S 0 in the growth media resulted in decreases in specific activities of two cytoplasmic hydrogenases (I and II) and of a membrane-bound hydrogenase, each by an order of magnitude. The primary S 0 -reducing enzyme in this organism and the mechanism of the S 0 dependence of peptide metabolism are not known. This study provides the first evidence for a highly regulated fermentation-based metabolism in P. furiosus and a significant regulatory role for elemental sulfur or its metabolites.Hyperthermophiles are microorganisms that grow optimally at 80°C and above (46,47). Virtually all of them are strict anaerobes, and most are heterotrophs. All of the heterotrophs utilize peptides as a carbon source, and most use elemental sulfur (S 0 ) as a terminal electron acceptor leading to H 2 S production. The most studied of the S 0 -reducing, heterotrophic hyperthermophiles are species of Pyrococcus. Most of these organisms only utilize peptide-related substrates as a carbon source and show no significant growth in the absence of S 0 (9,12,19,36). Notable exceptions are Pyrococcus furiosus, P. woesei, and P. glycovorans, which are capable of metabolizing poly-and oligosaccharides, as well as peptides (2, 4, 10). P. furiosus and P. woesei can also grow to high cell densities in the absence of S 0 . The pathways of peptide and carbohydrate metabolism have been well studied in P. furiosus (1, 7). Glycolysis appears to occur via a modified Embden-Meyerhof pathway (Fig. 1) (22,35). This pathway is unusual in that the hexose kinase and phosphofructokinase steps are dependent on ADP rather than ATP, and a novel tungsten-containing enzyme termed glyceraldehyde-3-phosphate:ferredoxin oxidoreductase (GAPOR) replaces the expected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase. Amino acid catabolism in P. furiosus is thought to involve four distinct 2-keto acid oxidoreductases that convert transaminated amino acids into their corresponding coenzyme A (CoA) derivatives (Fig. 2) (3,15,31,32). These CoA derivatives, together with acetylCoA produced from glycolysis via pyruvate...