V(D)J recombination, the process that assembles antigen-receptor genes, is directed by signal sequences flanking the DNA segments to be joined. Signals consist of a conserved heptamer and nonamer separated by a spacer of either 12 or 23 base pairs. Recombination occurs almost exclusively between two signals with spacers of different lengths. This restriction, called the '12/23 rule', governs the organization and pattern of rearrangement of antigen-receptor loci. In vitro work demonstrating the direct roles of the Rag proteins in the initiation of V(D)J recombination did not recreate the 12/23 rule. Instead, double-strand breaks were formed efficiently at isolated signals. Here we show that extracts made from a lymphoid cell line that expresses truncated forms of the Rag1 and Rag2 proteins have a signal-cutting activity that obeys the 12/23 rule. Cleavage at the two signals is concerted and requires their synapsis, and mutations of one signal prevent cleavage at both.