We have isolated a cDNA clone encoding a novel helixloophdix (HLH) protein, Id. Id is missing the basic region adjacent to the HLH domain that is essential for specific DNA binding in another HLH protein, MyoD. An in vitro translation product of Id can associate specifically with at least three HLH proteins (MyoD, E12, and E47) and attenuate their ability to bind DNA as homodlmeric or heterodimeric complexes. Id is expressed at varying levels in all cell lines tested. In three cell lines that can be induced to undergo terminal dlfferentlation, Id RNA levels decrease upon induction. Bansfection experiments indicate that overexpresston of Id inhibits the frans-activation of the muscle creatine klnase enhancer by MyoD. Based on these findings, we propose that HLH proteins lacking a baste region may negatively regulate other HLH proteins through the formation of nonfunctional heterodimerit complexes.