Rearrangement of DQ52 with JH in IgH genes takes place in a large part of lymphocytes at a very early stage of differentiation although usage of DQ52 seems to be limited in the functional IgH genes of mature B cells. A possible and likely explanation for this, is secondary D-JH joinings occurring after the initial DQ52-JH joinings, which becomes possible only when DQ52 segments join 5'-located JH segments. Previously we reported the non-random use of JH in joinings with DQ52, in which DQ52-JH2 joinings are dominant. We questioned how the preferential usage of JH2 in DQ52-JH joinings is determined by utilizing extrachromosomal DNA substrates. Extrachromosomal DNA substrates with a combination of DQ52 and multiple JH segments were prepared. These plasmids with a polyoma-derived replication system were transfected into pre-B cell lines and recombination profiles of recovered plasmids were analyzed semi-quantitatively. Experiments with plasmids containing all four JH segments in normal configuration showed an apparent high frequency of JH2 usage, similar to the rearrangement previously observed in thymocyte genomes. In plasmids containing two JH segments, when one was JH2, it was rearranged more frequently than the other, independent of its proximity to DQ52. The JH3 with recombination signal sequences (RSS) substituted for JH2 was rearranged more frequently than the naive JH2 itself indicating the importance of RSS. A JH3 hybrid with the 200 bp upstream sequence of JH2 but excluding RSS, however, failed to increase JH3 use. The addition of one nucleotide to the 22-bp spacer of JH1 RSS increased the frequency of JH1 usage. These observations suggest that the preferential usage of JH2 is determined by the primary DNA sequence and is largely dependent on RSS.