Adenosine triphosphatase (ATPase) fromThiobacilhis ferrooxidanswas purified 55-fold. Polyacrylamide gel electrophoresis of the most purified fraction showed only one major band; histochemical analysis showed that the ATPase activity was associated with this band. The pH optimum is 9–10. The enzyme hydrolyzed ATP stoichiometrically to ADP and inorganic phosphate, theKmfor this substrate being 7.75 × 10−3 M. GTP and ITP are alternate substrates, theKmvalues for these being 6.71 × 10−3 M and 3.12 × 10−3 M, respectively. ADP is slightly hydrolyzed. Magnesium, manganese, and calcium can serve as cofactors;Kmvalues for these are 2.0 × 10−3 M, 9.4 × 10−4 M, and 8.0 × 10−4 M, respectively. The enzyme activity was not activated by either sodium or potassium, but a combination of the two ions were inhibitory. Azide andp-hydroxymercuribenzoate strongly inhibited the enzyme activity, whereas cyanide, dinitrophenol, andN,N′-dicyclohexylcarbodiimide (DCCD) were without effect. The enzyme was cold labile at 0 °C, but was more stable at 18–24 °C.