The pituitary homeobox factor-1 (Pitx-1) transactivates a number of pituitary-specific genes through direct interaction with other specific transcription factors. We demonstrate here that Pitx-1 plays a crucial role in the regulation of the Chinook salmon luteinizing hormone beta gene promoter through a number of novel mechanisms. On the proximal promoter its action involves a synergistic effect with steroidogenic factor-1 (SF-1) alone or in combination with the estrogen receptor; promoter activity being induced by 9- or 35-fold over controls, respectively. Further upstream, a series of four Pitx-1 response elements (located between 1366 and 1506 bp from the transcriptional start site) is also involved in regulating the promoter activity. The two distal sequences have the greatest effect on the basal activity and are also essential for the gonadotropin-releasing hormone (GnRH) response. Mammalian two-hybrid assays revealed that Pitx-1 can homodimerize. Moreover, circular permutation assays indicate that binding of Pitx-1 to more than one response element induces conformational changes of the target DNA. This constitutes an additional mechanism through which Pitx-1 can mediate transactivation of this gene, allowing the demonstrated interaction of proximal response elements and distal enhancers, thus facilitating the maximal GnRH response that was seen in the longer promoter constructs. Our research also indicates that Pitx-1 is phosphorylated on three residues when bound to the DNA.