An active pyrophosphate-dependent 6-phosphofructokinase (PPi-PFK) from the thermotolerant methanotroph Methylococcus capsulatus Bath, containing a six-residue polyhistidine tag, was characterized. The enzyme was homodimeric (2 × 45 kDa), nonallosteric and most active at pH 7.0. PPi-PFK catalyzed reactions of PPi-dependent phosphorylation of fructose-6-phosphate (F-6-P) (Km 2.27 mM and Vmax 7.6 U mg-1 of protein), sedoheptulose-7-phosphate (Km 0.027 mM and Vmax 31 U mg-1) and ribulose-5-phosphate. In the reaction with F-6-P, the apparent Km for PPi was 0.027 mM, while in the reverse reaction, Km for orthophosphate was 8.69 mM and that for fructose-1,6-bisphosphate 0.328 mM (Vmax 9.0 U mg-1). Phylogenetically, M. capsulatus PPi-PFK was most similar to PPi-PFKs from the lithoautotrophic ammonia oxidizers Nitrosomonas europaea (74.0%), Nitrosospira multiformis (73.6%) and Betaproteobacterial methylotroph Methylibium petroleiphilum PM1 (71.6% identity). Genes coding PPi-PFK and a putative V-type H+-translocating pyrophosphatase (H+-PPi-ase) were cotranscribed as an operon. The potential significance of the PPi-PFK for regulation of carbon and energy fluxes in M. capsulatus Bath is discussed.