Matrix-associated regions (MARs), AT-rich DNA segments that have an affinity for the nuclear matrix, have been shown to play a role in transcriptional regulation of eukaryotic genes. The present study demonstrates that a DNA element, called L2a, which has been implicated in the transcriptional regulation of the mouse CD8a gene encoding an important T cell coreceptor, is a MAR. Moreover, the identities of two nuclear proteins, L2a-P1 and L2a-P2, previously shown to bind to the L2a element, have been determined. The L2a-P1 protein found to be present in all CD8-positive T cell lines tested is SATB1, a known MAR-binding protein. The widely expressed L2a-P2 protein is CDP/Cux, a MAR-binding protein that has been associated with repression of gene transcription. Interaction of both proteins with the L2a element was studied using the missing nucleoside approach, DNase I footprinting, and electrophoretic mobility shift assays with wild type and mutant L2a elements. The data suggest that CDP/Cux bound to the L2a element is displaced by binding of SATB1 and the accompanying conformational change in the DNA lying between the primary binding sites of SATB1 and CDP/Cux. We suggest that displacement of CDP/Cux by SATB1 favors transcription of the CD8a gene, possibly by enhancing or altering its association with the nuclear matrix.