Replication factor C (RF-C), a complex of five subunits, and several subassemblies of RF-C, representing intermediates along the proposed protein assembly pathway (Podust, V. N., and Fanning, E. (1997) J. Biol. Chem. 272, 6303-6310), were expressed in insect cells using baculoviruses encoding individual subunits (p140, p40, p38, p37, and p36). Purified proteins were analyzed for ATPase activity to assess the role of individual subunits in ATP hydrolysis. His-tagged p40 contained low ATPase activity, but tagged p37 and p36 did not. Complexes of p40.p37.p36 bearing a His tag on any subunit displayed DNA-stimulated ATPase activity, in agreement with a recent report (Cai, J., Gibbs, E., Uhlmann, F., Philips, B., Yao, N., O'Donnell, M. , and Hurwitz, J. (1997) J. Biol. Chem. 272, 18974-18981). In contrast, complex p38.p37.p36-his displayed no ATPase, suggesting that p40 is essential for ATPase activity. Although p38 was not required for ATPase activity, the activity of the p40-his.p38.p37. p36 complex was more salt-resistant than that of the p40-his.p37.p36 complex. The p140 subunit further increased the specific ATPase activity of RF-C complex by enhancing its stimulation by DNA. Taken together, the data indicate that all five RF-C subunits constitute ATPase activity, although the contributions of the individual subunits differ. Predicted ATP-binding domains of all five subunits were mutated to assess the importance of multiple ATP-binding sites of RF-C. In each case, the Lys of the conserved P-loop motif was replaced by Glu. The ATP-binding domain of p38 was found to be dispensable for the activity of the five-subunit RF-C in polymerase delta DNA synthesis. In contrast, mutation of the ATP-binding domains in other RF-C subunits impaired RF-C assembly, function, or both.