A hyperthermophilic alkaline phosphatase was purified from Thermotoga neapolitana by heat treatment at 100 degrees C in the presence of Co2+ followed by ion-exchange and affinity chromatographies. The enzyme was purified 2,880-fold with 44% yield. The purified enzyme showed a single protein band of M-r 45,000 on SDS-PAGE and an apparent molecular weight of 87,000 estimated by gel filtration chromatography. This suggested a homogenous dimer structure. The optimal pH and temperature for enzyme activity were 9.9 and 85 degrees C, respectively. Under optimal conditions, T. neapolitana alkaline phosphatase displayed 30% higher activity than calf intestine alkaline phosphatase did with p-nitrophenyl-phosphate as substrate. The hyperthermostable enzyme had a half-life of 238 min at 90 degrees C and K-m and V-max values of 183 mu M and 1,352 U mg(-1), respectively. Co2+ enhanced the enzyme activity, thermostability, and ligand affinity during column chromatography. The alkaline phosphatase was twice as active with Co2+ than with either Zn2+ or Mn2+ as the metal cofactor.