The proteins RAG-1 and RAG-2 are essential for initiation of V(D)J recombination. In dividing cells, RAG-2 accumulates during G1 and is undetectable during the S and G2/M cell cycle phases. A conserved degradation signal, including an essential CDK phosphorylation site at Thr-490, regulates RAG-2 accumulation during cell division and links V(D)J recombination to the cell cycle. Mutations within this signal abolish periodic degradation of RAG-2 protein in dividing cells. In mice expressing endogenous or wild-type transgenic RAG-2, V(D)J recombination intermediates accumulate preferentially in GO/G1 thymocytes; this restriction is relieved by mutation of Thr-490 to alanine (T490A). Thus, periodic destruction of RAG-2 protein couples V(D)J recombination to cell cycle phase. Using transgenic mice expressing the T490A RAG-2 mutant and a functional T cell receptor β chain, we demonstrate that coupling of V(D)J recombination to the cell cycle is not essential for enforcement of allelic exclusion.