Functional variable (V), diversity (D), and joining (J) gene segments contribute unequally to the primary repertoire. One factor contributing to this nonrandom usage is the relative frequency with which the different gene segments rearrange. Variation from the consensus sequence in the heptamer and nonamer of the recombination signal sequence (RSS) is therefore considered a major factor affecting the relative representation of gene segments in the primary repertoire. In this study, we show that the sequence of the spacer is also a determinant factor contributing to the frequency of rearrangement. Moreover, the effect of the spacer on recombination rates of various human Vkappa gene segments in vitro correlates with their frequency of rearrangement in vivo in pre-B cells and with their representation in the peripheral repertoire.