Pit1, pituitary-specific transcription factor 1, regulates differentiation of cells of the Pit1 lineage in the anterior pituitary and the synthesis of peptide hormones by these cell types, including GH. Pit1 is characterized by an N-terminal transactivation domain and a C-terminal POU domain. Alternative forms of Pit1, differing from each other in the N-terminal domain, have been reported in several species, but the functional implication of having multiple isoforms is not known. Several PIT1 mRNA transcripts exist in chickens that have not been characterized. This study was conducted to determine which, if any, of the chicken Pit1 isoforms regulate the chicken GH (cGH) promoter. During the course of this work, Pit1β2, a novel isoform of chicken Pit1, was discovered. Effects of known and novel isoforms (Pit1α, Pit1β1, Pit1β2, and Pit1γ) on cGH promoter activity were characterized in chicken Leghorn male hepatoma cells. Three of the isoforms, Pit1α, Pit1β1, and Pit1β2, activated the cGH promoter, whereas Pit1γ did not. Results from gel-shift assays indicated that Pit1γ does not bind to the proximal Pit1-bindng site of the cGH promoter, suggesting a possible mechanism underlying its inactivity. We found a functional advantage for having multiple isoforms expressed. When Pit1β1 was coexpressed with Pit1α or Pit1β2, significantly greater activation of the cGH promoter occurred than with any one isoform alone, with synergistic activation occurring when Pit1α and Pit1β1 were coexpressed. Whether this increased activation required, or was facilitated by, heterodimerization of two isoforms is not known. Identification of isoforms with specific functions will facilitate identification of their respective interacting partners that are essential for GH gene expression.