It has been proposed that the helix-loop-helix (HLH) protein Id serves as a general antagonist of cell differentiation by inhibiting bHLH (HLH with an adjacent stretch of basic amino acids) proteins specifically required for developmental programs (such as MyoD). We show here that ectopic expression of Id represses in vivo activity of the bHLH protein E2-5 (encoded by the E2A gene) and of both the immunoglobulin heavy-chain (IgH) and kappa-light-chain gene enhancers to which E2-5 binds. Id does not affect the activity of the bHLH-zip protein, TFE3, which also binds these enhancers. We examined a large panel of B-cell lines that represent different stages of lymphoid development and found only two that express Id mRNA. The cell lines Ba/F3 and LyD9 have been categorized previously as early B-lymphoid-cell progenitors. Unlike their more mature B-lymphoid-cell counterparts, Ba/F3 and LyD9 cells do not express I mu sterile transcripts, which are indicative of IgH enhancer activity. Moreover, Ba/F3-derived nuclear extracts lack E2-box-binding activity, indicating the absence of free bHLH proteins, and transfected Ba/F3 cells fail to support the activity of the IgH enhancer. Hence, expression of Id correlates inversely with bHLH protein activity and enhancer function in vivo. These results suggest that Id may play a role early in B-lymphoid-cell development to regulate transcription of the IgH locus.