USF1, a basic region/helix-loop-helix/leucine zipper (bHLHZ) transcription factor, binds to the E-box in the PAI-1 (plasminogen activator inhibitor) promoter. Two alleles containing the E-box control PAI-1 transcription; these alleles are termed "4G" and "5G" based on the G tract flanking E-box. USF1-governed transcription of PAI-1 is elevated in heritable asthma sufferers: the 4G/4G genotype has the highest plasma levels of PAI-1. While USF1 uses its basic region to bind E-box, we found that it uses its 12 amino-acid loop to recognize the flanking sequence and discern the single-nucleotide difference between the alleles. We used the bacterial one-hybrid and electrophoretic mobility shift assays to assess protein-DNA recognition, and circular dichroism to examine protein secondary structure. We mutated Ser233 and Thr234 in the USF1 bHLHZ loop to Ala to generate S233A and T234A. Interestingly, USF1 bHLHZ, S233A, and T234A prefer the 5G sequence (USF1 bHLHZ K-d values 4.1 +/- 0.3 nM and 7.0 +/- 0.4 nM for SG and 4G, respectively), whereas studies in stimulated human mast cells showed a preference for 4G. We replaced the 8 amino-acid loop of transcription factor Max bHLHZ with the 12 amino-acid USF1 loop: this mutant now distinguishes the 4G/5G polymorphism-while Max bHLHZ does not-confirming that USF1 differentiation of the 4G/5G is driven by the loop.