The serine proteinase inhibitor (SPI-3) gene expression is transcriptionally regulated by interleukin (IL)-6 and glucocorticoids in hepatic cells. To identify the transcription factors involved in regulation of the SPI-3 promoter-chloramphenicol acetyltransferase constructs we overexpressed Signal Transducer and Activator of Transcription (STAT) proteins (STAT1, STAT3, STAT5B, and STAT6) and CAAT enhancer-binding protein beta. Specific signaling pathways were activated by cointroduced receptors for growth hormone, IL-3, IL-4, or chimeric receptors containing the cytoplasmic domain of gp130. STAT3 and STAT5B induced transcription via the SPI-3 promoter. The STAT5B response was substantially enhanced by truncation of the 5'-flanking region from -1021 to -148. The responsiveness to STAT3 and STAT5B required the STAT binding element at -132 to -124. This element was sufficient to confer regulation onto a heterologous promoter gene construct. In contrast, overexpression of CAAT enhancer-binding protein beta reduced the transcriptional activity of the SPI-3 promoter, presumably by interfering with STAT protein binding to the promoter element. The SPI-3 gene is the first example of an acute phase gene that is responsive to both STAT3 and STAT5B.