Nucleoside diphoshate kinases (NDKs), an evolutionarily conserved family of proteins, synthesize nucleoside triphosphates from nucleoside diphosphates and ATP. Here, we have characterized the kinase activity and DNA processing functions of eight human proteins that contain at least one domain homologous to Escherichia coli NDK. Not all human proteins with NDK-like domains exhibited NDK activity when expressed as recombinant proteins in E. coli. Human NDK1 (NM23-H1) has been reported to have 3′ → 5′ exonuclease activity. In addition to human NDK1, we also find that human NDK5, NDK7, and NDK8 contain 3′ → 5′ exonuclease activity. Site-directed mutagenesis, competition assays between wild-type and mutant NDK proteins, and NMR studies confirmed that the DNA-binding and 3′ → 5′ exonuclease activity of human NDK1 is an intrinsic activity of the protein. Using double-stranded DNA substrates containing modified bases, human NDK1 efficiently excised nucleotides from the single-strand break produced by APE1 or Nth1. When human cells were treated with various DNA-damaging agents, human NDK1 translocated from the cytoplasm to the nucleus. These results suggest that, in addition to maintenance of nucleotide pool balance, the human NDK-like proteins may have previously unrecognized roles in DNA nucleolytic processing. © 2005 American Chemical Society.