STAT4, which plays a pivotal role in Th1 immune responses, enhances IFN-gamma transcription in response to the interaction of IL-12 with the IL-12R. Mice deficient in STAT4 lack IL-12-induced IFN-gamma production and Th1 differentiation and display a predominantly Th2 phenotype. Although these findings indicate that STAT4 expression levels are important for the development of cytokine-producing Th1 cells, the transcriptional and posttranscriptional mechanisms regulating STAT4 expression are unknown. We sought to identify and characterize the transcriptional regulatory elements in the promoter region of the human STAT4 gene. We found that disruption of multiple transcriptional regions covering the CREB, OCT1, and SP1 motifs significantly reduced STAT4 promoter activity. However, genomic DNA isolated from 91 patients with asthma or rheumatoid arthritis showed no evidence of mutations in the defined STAT4 essential promoter region. The 5' flanking region of the promoter was found to contain a -149A/G change in approximately 20-35% of patients, but this polymorphism had no effect on promoter activity. Interestingly, STAT4 expression was drastically increased in human T cells following treatment with a DNA methyltransferase inhibitor, and truncation of methylation sites in the proximal regulatory elements of the STAT4 promoter markedly enhanced transcriptional activity. Thus, our findings provide molecular insight into STAT4 expression and suggest that, in human T cells, STAT4 expressional regulation is associated with DNA hypermethylation, but not promoter polymorphisms.