Allelic exclusion is the process wherein lymphocytes express Ag receptors from only one of two possible alleles, and is effected through a feedback inhibition of further rearrangement of the second allele. The feedback signal is thought to cause chromatin changes that block accessibility of the second allele to the recombinase. To identify the putative chromatin changes associated with allelic exclusion, we assayed for DNase I hypersensitivity, DNA methylation, and transcription in 100 kb of the TCR-beta locus. Contrary to current models, we identified chromatin changes indicative of an active and accessible locus associated with the occurrence of allelic exclusion. Of 11 DNase I hypersensitive sites identified, 3 were induced during CD4-CD8- to CD4+CD8+ thymocyte differentiation, and demethylation and increased germline transcription of the locus were evident. We further examined the role of the most prominently induced site near the TCR-beta enhancer (E beta) in allelic exclusion by targeted mutagenesis. Two other sites were also examined in New Zealand White (NZW) mice that have a natural deletion in the TCR-beta locus. TCR-beta gene recombination and allelic exclusion were normal in both mutant mice, negating dominant roles for the three hypersensitive sites in the control of allelic exclusion. The data suggest that alternative cis-regulatory elements, perhaps contained in the E beta enhancer and/or in the upstream V beta region, are involved in the control of TCR-beta allelic exclusion.