Human cytomegalovirus (HCMV) terminase is composed of subunits pUL56 (130 kDa) and pUL89 ( approximately 75 kDa), encoded by the UL56 and UL89 genes. In a recent investigation, we demonstrated that the main ATPase activity is associated with the large terminase subunit pUL56. The protein has two putative ATP-binding sites, which were suggested to be composed of the sequence (amino acids 463-470) for ATP-binding site 1 and YNETFGKQ (amino acids 709-716) for the second site. We now demonstrate using a 1.5 kb fragment encoding the C-terminal half of pUL56 that ATP-binding site 1 is not critical for the function, whereas ATP-binding site 2 is required for the enzymatic activity. Mutation G714A in this protein reduced the ATPase activity to approximately 65% and the double mutation G714A/K715N showed a reduction up to 75%. However, the substitution of E711A revoked the effect of the substitutions. The functional character of the ATP-binding site was demonstrated by transfer of YNETFGKQLSIACLR (709-723) to glutathione-S-transferase (GST). Interestingly, vanadate, an ATPase inhibitor, has the ability to block the ATPase activity of pUL56 as well as of Apyrase, while the antitumor ATP-mimetic agent geldanamycin, did not affect the ATP-binding of pUL56. Furthermore, in contrast to an inactive control compound, the specific HCMV terminase inhibitor BDCRB showed a partial inhibition of the pUL56-specific ATPase activity. Our results clearly demonstrated that (i) the enzymatic activity of the terminase subunit pUL56 could be inhibited by vanadate, (ii) only the ATP-binding site 2 is critical for the pUL56 function and (iii) glycine G714 is an invariant amino acid.