Pit-1 is a pituitary-specific transcription factor responsible for activating growth hormone (GH) and prolactin genes. Here, we describe the isolation of a rainbow trout cDNA clone that contains the entire Pit-1 coding region. The deduced amino acid sequence contains 358 residues, encoding a 39-kDa protein. Comparison of the protein sequences of the rainbow trout and rat Pit-1 shows that the 160 residue POU domain at the C terminus is highly conserved (86% identical). However, homology is much weaker in the N-terminal region (56% identical), and the rainbow trout Pit-1 contains segments of 29 and 33 amino acids that are not present in rat Pit-1. The protein produced by expression of rainbow trout Pit-1 cDNA in Escherichia coli binds specifically to at least four sites in the rainbow trout GH gene promoter. Moreover, we demonstrate that the promoter region of salmon somatolactin gene, which belongs to the GH prolactin gene family and is also expressed specifically in the pituitary, has at least five rainbow trout Pit-1 binding sites. The consensus sequence of these binding sites closely matches the 9-base pair motif, (T/A)(T/A)TATNCAT, recognized by rat Pit-1. Rainbow trout Pit-1 specifically activates rainbow trout GH promoter fusion gene expression, confirming the ability of Pit-1 to bind in a transcriptionally active conformation in GH gene promoter.