Tissue-specific expression of the murine TCR/CD3-delta gene is regulated by multiple factors. Earlier, we reported that this gene has a tissue-specific enhancer at its 3' end that consists of two cis-acting elements, M delta A and M delta B. This study demonstrates that at least two independent factors bind to the 22 nucleotide long M delta A element. A T cell-specific factor termed M delta AF recognizes a 10-bp sequence TTCCATGACA, whereas the cAMP response element binding protein (CREB) recognizes an overlapping sequence TGACATCA. In vitro studies indicated that the 135-kDa M delta AF and the 43-kDa CREB competed for binding to the M delta A element. To test this competition in vivo, murine T cells (EL4) were cotransfected with a CREB expression plasmid and a reporter plasmid containing the M delta A element. The presence of an excess of CREB protein in the cell inhibited the CAT activity completely. This inhibitory effect of CREB could be overcome by cotransfection of an additional vector containing the somatostatin CRE site that prevented the overexpressed CREB protein from binding to the M delta A element. It is possible that the murine CD3-delta gene in T cells is regulated by the relative levels of the T cell-specific DNA binding protein M delta AF and the ubiquitous CREB in conjunction with other factors.