Eukaryotic ssDNA viruses encode a rolling-circle replication (RCR) initiation protein, Rep, which binds to iterated DNA elements functioning as essential elements for virus-specific replication. By using the iterons of all known circoviruses, nanoviruses and nanovirus-like satellites as heuristic devices, we have identified certain amino acid residues that presumably determine the DNA-binding specificity of their Rep proteins. These putative "specificity determinants" (SPDs) cluster in two discrete protein regions, which are adjacent to distinct conserved motifs. A comparable distribution of SPDs was uncovered in the Rep protein of geminiviruses. Modeling of the tertiary structure of diverse Rep proteins showed that SPD regions interact to form a small beta-sheet element that has been proposed to be critical for high-affinity DNA-binding of Rep. Our findings indicate that eukaryotic circular ssDNA viruses have a common ancestor and suggest that SPDs present in replication initiators from a huge variety of viral and plasmid RCR systems are associated with the same conserved motifs.