Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of phi29 DNA polymerase. The affinity for nucleotides of phi29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of phi29 DNA polymerase. The results suggest that Lys371 of phi29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that phi29 DNA polymerase can achieve the closed conformation in the absence of a DNA template.