The process of antigen receptor gene rearrangement, V(D)J recombination, involves DNA cleavage by the RAG-1 and RAG-2 proteins. Cleavage generates covalently sealed (hairpin) DNA ends, termed coding ends, which must be opened by an endonuclease prior to joining. Resolution of these hairpin ends requires the activity of the DNA-dependent protein kinase (DNA-PK), a protein kinase whose specific role is yet undetermined. It has been suggested that phosphorylation of one or both RAG proteins by DNA-PK is required to activate or recruit the hairpin-opening nuclease. Furthermore, very recent work has shown that RAG proteins themselves can open hairpins. These data raise the possibility that DNA-PK-mediated phosphorylation of the RAG proteins could regulate the hairpin opening reaction. To test this hypothesis, we constructed mutant versions of RAG-1 and RAG-2 in which all four DNA-PK consensus phosphorylation sites were removed by site-directed mutagenesis. Our data provide conclusive evidence that phosphorylation of these conserved serine/threonine residues is not required for hairpin opening or joining of V(D)J recombination intermediates.