The Escherichia coli DNA polymerase III tau and gamma subunits are single-strand DNA-dependent ATPases (the latter requires the delta and delta' subunits for significant ATPase activity) involved in loading processivity clamp beta. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic tau/gamma homologs and 1 eukaryotic tau/gamma homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.