V(D)J recombination is initiated by RAG1 and RAG2, which together with HMGB1 bind to a recombination signal sequence (12RSS or 23RSS) to form the signal complex (SC) and then capture a complementary partner RSS, yielding the paired complex (PC). Little is known regarding the structural changes that accompany the SC to PC transition or the structural features that allow RAG to distinguish its two asymmetric substrates. To address these issues, we analyzed the structure of the 12RSS in the SC and PC using fluorescence resonance energy transfer (FRET) and molecular dynamics modeling. The resulting models indicate that the 12RSS adopts a strongly bent V-shaped structure upon RAG/HMGB1 binding and reveal structural differences, particularly near the heptamer, between the 12RSS in the SC and PC. Comparison of models of the 12RSS and 23RSS in the PC reveals broadly similar shapes but a distinct number and location of DNA bends as well as a smaller central cavity for the 12RSS. These findings provide the most detailed view yet of the 12RSS in RAG–DNA complexes and highlight structural features of the RSS that might underlie activation of RAG-mediated cleavage and substrate asymmetry important for the 12/23 rule of V(D)J recombination.