Dihydroorotate dehydrogenase (DHOD) (EC 1.3.3.1) from the thermoacidophilic archaeon Sulfolobus solfataricus P2 (DSM 1617) was partially purified 3,158-fold, characterized, and the encoding genes identified. Based on enzymological as well as phylogenetic methods, dihydroorotate dehydrogenase from S. solfataricus (DHODS) represents a new type of DHOD, type 1S. Furthermore, it is unable to use any of the (type-specific) natural electron acceptors employed by all other presently known DHODs. DHODS shows optimal activity at 70°C in the pH range 7-8.5. It is capable of using ferricyanide, 2,6-dichlorophenolindophenol (DCIP), Q₀, and molecular oxygen as electron acceptor. Kinetic studies employing ferricyanide indicate a two-site ping-pong mechanism with K_M values of 44.2-1.9 µM for the substrate dihydroorotate and 344-21 µM for the electron acceptor ferricyanide, as well as competitive product inhibition with a K_i of 23.7-3.4 µM for the product orotate (OA). The specific activity, as determined from a partially purified sample, is approximately 20 µmol mg^-1 min^-1. DHODS is a heteromeric enzyme comprising a catalytic subunit encoded by pyrD (291 aa; MW=31.1 kDa) and an electron acceptor subunit (208 aa; MW=23.6 kDa), encoded by orf1. DHODS employs a serine as catalytic base, which is unique for a cytosolic DHOD. To our knowledge, this work represents not only the first study on an archaeal DHOD but the first on a nonmesophilic DHOD as well.