We have generated mutant mice in which TCR beta chain enhancer (E-beta) was replaced with the TCR beta chain enhancer (E-alpha). Using this mouse model, we analyzed (1) recombination status of the TCR beta chain genes after functional V(D)J rearrangements occurred in the first allele during double-negative (DN)-to-double-positive (DP) transition and (ii) involvement of E-beta for the expression of rearranged TCR beta chain genes. Our data show that E-alpha substituted for E-beta function to express a similar extent of TCR beta chains exactly at the same time as did E-beta (CD25(+)CD44(-) DN stage), although the proportion of TCR beta (+) cells at this stage was low in mutant mice. At the DP stage, germline transcription and histone acetylation of D-beta-J(beta) loci were detectable at a high degree in both mutant and wild-type mice. However, DP cells in mutant mice retained the germline D-beta-J(beta) configuration at a higher frequency than that of wild-type mice, whereas both DP cells expressed TCR beta chains to a similar extent. These data suggest that chromatin opening has a limited impact on D-beta-to-J(beta) recombination at the DP stage and that E-alpha is functionally equivalent to E-beta in promoting expression of functionally rearranged TCR beta chain genes through DN-to-DP transition.