Replication factor C (RFC) is a five-subunit protein complex required for coordinate leading and lagging strand DNA synthesis during S phase and DNA repair in eukaryotic cells. It functions to load the proliferating cell nuclear antigen (PCNA), a processivity factor for polymerases delta and epsilon, onto primed DNA templates. This process, which is ATP-dependent, is carried out by 1) recognition of the primer terminus by RFC () binding to and disruption of the PCNA trimer, and then 3) topologically linking the PCNA to the DNA. In this report, we describe the purification and properties of recombinant human RFC expressed in Sf9 cells from baculovirus expression vectors. Like native RFC derived from 293 cells, recombinant RFC was found to support SV40 DNA synthesis and polymerase delta DNA synthesis in vitro and to possess an ATPase activity that was highly stimulated by DNA and further augmented by PCNA. Assembly of RFC was observed to involve distinct subunit interactions in which both the 36- and 38-kDa subunits interacted with the 37-kDa subunit, and the 40-kDa subunit interacted with the 36-kDa subunit-37-kDa subunit subcomplex. The 140-kDa subunit was found to require interactions primarily with the 38- and 40-kDa subunits for incorporation into the complex. In addition, a stable subcomplex lacking the 140-kDa subunit, although defective for DNA replication, was found to possess DNA-dependent ATPase activity that was not responsive to the addition of PCNA.