Myogenic factors and TAL1 possess distinguishable DNA binding characteristics when they form a complex with basic helix-loop-helix (bHLH) proteins of class A. These characteristics were evident in electrophoretic mobility shift assays showing that complexes of myogenic factors and HTF4 displayed a relatively high affinity for the enhancer in the muscle creatine kinase gene, whereas TAL1 appeared to greatly attenuate the interaction of HTF4 with this enhancer. In addition, by forming a complex with HTF4 in solution, TAL1 could exert a negative effect on the interactions of HTF4 with elements that include E box motifs of microE2 (CAGCTG) and kappa E2/microE5 (CACCTG) type. Similarly, heterodimers containing TAL1 and the DNA binding domain of E47 exhibited a relatively weak affinity for microE2 and kappa E2/microE5 core motifs. The results of both studies invoked the hypothesis that in vivo TAL1 might act as a negative regulator of microE2 and kappa E2/microE5 sequence motifs by forming a complex with the products of the E2A and HTF4 genes. Support for this hypothesis was obtained by transient expression analyses where TAL1 was found to inhibit the activation effects produced by E2-5 and HTF4a on a reporter gene construct containing repeated microE2 and microE5 motifs, derived from the immunoglobulin gene enhancer.