Using transgenic substrates, we found that the immunoglobulin κ gene 3′ enhancer (E3′) acts as a negative regulator in Vκ-Jκ joining. Although the E3′ was originally identified as a transcriptional enhancer, it acts in a Buppressive manner for recombinational regulation. Base substitution analysis has shown that the PU. 1-binding site within the E3′ regulates the B/T specificity of Vκ-Jκ joining. In a substrate with a mutated PU. 1-binding site (GAGGAA to TCTTCG), Vκ-Jκ joining occurred not only in B cells, but also in T cells. The E3′ region is also responsible for determining the pro-B/pre-B specificity of Vκ-Jκ joining. When the E3′ region was deleted, κ gene rearrangement actively occurred at the early pro-B stage of B cell development: non-germline (N) nucleotides were common at recombination junctions.