We isolated full-length cDNAs encoding the 43-kD form of human upstream stimulatory factor (USF), a cellular factor required for efficient transcription of the adenovirus major late (AdML) promoter in vitro. Sequence analysis showed USF to be a member of the c-myc-related family of DNA-binding proteins. Using proteins translated in vitro, we identified a DNA-binding domain near the carboxyl terminus, which includes both a helix-loop-helix motif and a leucine repeat. We show that USF interacts with its target DNA as a dimer. The leucine repeat is required for efficient DNA binding of the intact protein and for interactions between full-length and truncated USF proteins. Interestingly, it is not required for DNA binding of the isolated helix-loop-helix domain. The structure of different cDNA clones indicates that USF RNA is differentially spliced, and alternative exon usage may regulate the levels of functional USF protein.