The POU2F1 gene, which plays an important role in regulating the mammalian genome and development, has both a ubiquitous (U) and a tissue-specific (L) promoter and is subject to intricate regulation. Regions of POU2F1 gene were found to contain multiple binding sites for its product POU2F1 (Oct-1), a transcription factor. Interspecies homology in these regions was found to exceed 90% among the human, mouse, rat, pig, and dog genomes, almost all of the Oct-1 binding sites being identical. Some of the sites cluster in the vicinity of each of the two alternative promoters, while others are in the 5' noncoding region 6 kb upstream of the transcription start site. The presence of Oct-1 at the sites was demonstrated by chromatin immunoprecipitation and the electrophoretic mobility shift assay (EMSA). A POU2F1 knockdown activated the U promoter and downregulated the L promoter in Namalwa cells, while Oct-1 overexpression exerted an opposite effect. Thus, Oct-1 acts via negative feedback to autoregulate the U promoter through low-affinity Oct-1 binding sites and positive feedback to autoregulate the L promoter through high-affinity canonical (oct) sites when increasing in concentration in a natural context.