Activator 1 (A1; also called replication factor C), in conjunction with proliferating-cell nuclear antigen (PCNA), is essential for the elongation of primed DNA templates by DNA polymerases delta and epsilon. A1 contains five distinct subunits of 145, 40, 38, 37, and 36.5 kDa. Here we describe the isolation, sequence, and bacterial expression of a cDNA coding for the 40-kDa subunit. In keeping with the presence of an ATP-binding motif, the bacterially expressed 40-kDa subunit binds ATP. The interaction between the 40-kDa subunit and ATP was reduced by the addition of PCNA. In addition, antibodies raised against the 40-kDa subunit abolished the A1- and PCNA-dependent synthesis of DNA catalyzed by polymerase delta. The putative amino acid sequence of the 40-kDa subunit of A1 revealed significantly homology with the bacteriophage T4 gene 44 protein and, to a lesser degree, with the tau and gamma subunits of Escherichia coli DNA polymerase III holoenzyme.