We have previously reported that the majority of the archaea utilize a novel pathway for coenzyme A biosynthesis (CoA). Bacteria/eukaryotes commonly use pantothenate synthetase and pantothenate kinase to convert pantoate to 4'-phosphopantothenate. However, in the hyperthermophilic archaeon Thermococcus kodakarensis, two novel enzymes specific to the archaea, pantoate kinase and phosphopantothenate synthetase, are responsible for this conversion. Here, we examined the enzymatic properties of the archaeal phosphopantothenate synthetase, which catalyzes the ATP-dependent condensation of 4-phosphopantoate and beta-alanine. The activation energy of the phosphopantothenate synthetase reaction was 82.3 kJ mol(-1). In terms of substrate specificity toward nucleoside triphosphates, the enzyme displayed a strict preference for ATP. Among several amine substrates, activity was detected with beta-alanine, but not with gamma-aminobutyrate, glycine nor aspartate. The phosphopantothenate synthetase reaction followed Michaelis-Menten kinetics toward beta-alanine, whereas substrate inhibition was observed with 4-phosphopantoate and ATP. Feedback inhibition by CoA/acetyl-CoA and product inhibition by 4'-phosphopantothenate were not observed. By contrast, the other archaeal enzyme pantoate kinase displayed product inhibition by 4-phosphopantoate in a non-competitive manner. Based on our results, we discuss the regulation of CoA biosynthesis in the archaea.