The lymphoid-specific immunoglobulin mu heavy chain gene intron enhancer (muE) contains multiple binding sites for trans-acting nuclear factors. We have used a combination of in vitro and in vivo assays to reconstruct protein-DNA interactions on a minimal B cell-specific mu enhancer that contains three motifs, muA, muB, and muE3. Using ETS-domain proteins that transactivate the minimal enhancer in non-lymphoid cells, we show that (i) PU.1 binds coordinately to both muA and muB sites in vitro and (ii) in the presence of Ets-1, this factor binds to the muA site and PU.1 to the muB site. Two factors, TFE3 and USF, bind to the muE3 element. When the ETS proteins are present together with muE3 binding proteins, a three-protein-DNA complex is generated. Furthermore, we provide evidence for protein-protein interactions between Ets-1 and PU.1 proteins that bind to muA and muB sites, and between Ets-1 and TFE3 bound to the muA and mu3 sites. We propose that this domain of the mu enhancer is assembled into a nucleoprotein complex that contains two tissue-restricted ETS domain proteins that recognize DNA from the same side of the helix and one ubiquitously expressed bHLH-leucine zipper protein that binds between them, recognizing its site from a different side of the helix.