V(D)J recombination is targeted by recombination signal sequences (RSS) located immediately adjacent to immune receptor gene segments. While the RSS flanking D(H) segments appear to be equivalent, they are not randomly utilized. During D(H) to J(H) rearrangement, the 3' D(H) RSS is virtually exclusively utilized, suggesting that the 3' D(H) RSS could simply be a better target for the recombinase. However, when we examined V(H) to D(H) (without J(H)) rearrangements, we found that the preference for D(H) RSS use changes, so that the 5' D(H) RSS are preferred. This suggests that the 3' D(H) RSS are not simply superior targets for the V(D)J recombinase, but instead that certain 12/23-bp spacer RSS combinations work better together to target recombination than do others. We have analyzed a series of artificial recombination substrates to delineate cis sequences that affect D(H) RSS selection. Our data suggest that coding sequences adjacent to the D(H) RSS, flanking sequences outside the D(H) gene segment itself, and recombination partner all affect D(H) RSS targeting.