To elucidate mechanisms that regulate ordered and tissue-specific assembly of Ig and TCR variable region gene segments, we have introduced a recombination substrate comprised of germline TCR β V, D, and J gene segments into an Abelson murine leukemia virus-transformed pre-B cell line that actively rearranges endogenous Ig H chain variable region gene segments but does not rearrange endogenous light chain or TCR variable region gene segments. We find that these cells efficiently joln D β segments to J β segments within the mini-locus, but that they do not make any detectable site-specific rearrangements of the Introduced V β segment even though it is closely linked in the same construct to the D β . These findings suggest that factors necessary for V β to (D β )J B joining may be absent in these pre-B cells and also imply that the order in which TCR V β , D β , and J β segments are rearranged can be Influenced by factors other than the 12/23 recombination rule. Furthermore, in agreement wlth the an accessibility model of VDJ recombinase control, the D β region d the construct was found to be relatively more sensitive to DNAase I digestion in isolated nuclei when compared to the unrearranged V β region.