A growth hormone-inducible nuclear factor complex (GHINF), affinity- purified using the growth hormone response element (GHRE) from the promoter of rat serine protease inhibitor 2.1, was found to contain Stat5a and -5b, as well as additional components. The ubiquitous transcription factor yin-yang 1 (YY1) is present in GHINF. An antibody to YY1 inhibited the formation of the GHINF. GHRE complex in an electrophoretic mobility shift assay. Furthermore, Stat5 was co-immunoprecipitated from rat hepatic nuclear extracts with antibodies to YY1. An examination of the GHRE shows that, in addition to two γ-activated sites, it contains a putative YY1 binding site between the two γ-activated sites, overlapping them both. Mutation of this putative YY1 site results in a decrease of GHINF·GHRE complex formation in an electrophoretic mobility shift assay and a corresponding decrease in growth hormone (GH) response in functional assays. The glucocorticoid receptor was also present in GHINF, and Stat5 co-immunoprecipitates with glucocorticoid receptor in hepatic nuclear extracts from rats treated with GH. GH activation of serine protease inhibitor 2.1 requires the unique sequence of the GHRE encompassing the recognition sites of several transcription factors, and the interaction of these factors enhances the assembly of the transcription complex.