A growth hormone (GH)-inducible nuclear factor (GHINF) from rat liver has been purified to near homogeneity. On SDS-polyacrylamide gel electrophoresis and UV-cross-linking, a major band of mass ~93 kDa and a minor band of ~70 kDa are detected in the purified fraction. DNase I footprinting using purified GHINF yields a protected region of -149/-115 on the rat serine protease inhibitor 2.1 (Spi 2.1) promoter encompassed within the growth hormone response element (GHRE). Mutational analysis demonstrated that GHINF binds synergistically to two γ-interferon-activated sites (GAS) within the GHRE, with the 3' element being the pivotal binding domain. Functional assays show that both GAS elements are necessary for full GH response. GHINF has no immunoreactivity with either a C-terminal Stat1 antibody or an N-terminal Stat3 antibody, while cross-reacting with a C-terminal Stat5 monoclonal antibody. GHINF will bind to two GAS elements from the Stat5 binding region of the β-casein gene. These studies indicate that GHINF is a Stat5-related factor binding synergistically to two GAS elements to activate Spi 2.1 transcription.