We have previously identified a DNase I-hypersensitive site in the T cell receptor beta locus, designated HS1, that is located 400 base pairs upstream of the transcriptional enhancer Ebeta and is induced during CD4(-)CD8(-) to CD4(+)CD8(+) thymocyte differentiation. Using electrophoretic mobility shift assays, we show that HS1 induction correlates with increased binding of two nuclear factors, Cux/CDP and SATB1, to a 170-base pair DNA sequence within HS1. Furthermore, we demonstrate that HS1 is a nuclear matrix attachment region, referred to as MARbeta. These findings demonstrate that an analogous organization of cis-regulatory elements in which a nuclear matrix attachment region is in close proximity to an enhancer is conserved in the immunoglobulin and T cell receptor loci. In addition, we show that MARbeta represses Ebeta-dependent reporter gene expression in transient transfection assays. However, the targeted deletion of MARbeta from the endogenous locus does not change T cell receptor beta gene transcription in developing T cells. These contrasting results suggest a potential pitfall of functional studies of nuclear matrix attachment regions outside of their natural chromosomal context.