DNA double-strand repair factors in the non-homologous end joining (NHEJ) pathway resolve DNA double-strand breaks introduced by the recombination-activating gene (RAG) proteins during V(D)J recombination of T and B lymphocyte receptor genes. Defective NHEJ and subsequent failure of V(D)J recombination leads to severe combined immunodeficiency disease (SCID). We originally linked T(-)B(-)NK(+) SCID in Athabascan-speaking Native Americans in the Southwestern US and Northwest Territories of Canada to chromosome 10. However, despite a common ancestry, the null mutation in the Artemis gene that we found to be causal in the SCID among the Navajo and Apache Indians was not present in the Dine Indians in the Northwest Territories. We now report a novel homozygous missense mutation (R776W) in RAG-1 in three children with T(-)B(-)NK(+) SCID from two related families of Athabascan-speaking Dine Indians in the Canadian Northwest Territories. As expected, we found no increased sensitivity to ionizing radiation in patient fibroblasts. The impaired activity of this RAG-1 mutant in V(D)J recombination was confirmed by the EGFP-based V(D)J recombination assays. Overexpression of wild type RAG-1 in patient fibroblasts complemented V(D)J recombination, with recovery of both coding and signal joint formation. Our results indicate that the novel R776W missense mutation in RAG-1 is causal in the T(-)B(-)NK(+) SCID phenotype in Athabascan-speaking Dine Indians from the Canadian Northwest Territories.