Pit-1 is a member of a family of transcription factors sharing two regions of homology: a highly conserved POU-specific (POUS) domain and a more divergent homeodomain (POUHD). Analysis of mutant Pit-1 proteins suggests that, while the POUHD is required and sufficient for low affinity DNA binding, the POUS domain is necessary for high affinity binding and accurate recognition of natural Pit-1 response elements. Pit-1 is monomeric in solution but associates as a dimer on its DNA response element, exhibiting DNA-dependent protein-protein interactions requiring the POUS domain. Analysis of alpha-helical domains and conserved structures in Pit-1 suggests that POU domain proteins interact with their DNA recognition sites differently than classic homeodomain proteins, with both the POUHD and the POUS domain contacting DNA. Transcriptional activity of Pit-1 on enhancer elements is conferred primarily by a Ser- and Thr-rich N-terminal region unrelated to other known transcription-activating motifs.