Analyzing the induction kinetics and promoter elements regulating the expression of the transcription factor Egr-1, we found elevated levels of Egr-1-encoding mRNA in synovial fibroblasts of rheumatoid arthritis (RA) patients when compared to controls. By contrast, synovial lymphocytes and macrophages do not show an elevated Egr-1 transcription. Therefore, the overexpression of Egr-1 may serve as a diagnostic marker to characterize synovial fibroblasts of RA patients. To study the regulatory mechanisms controlling Egr-1 expression we analyzed the function of transcription factor binding sites located in the Egr-1 promoter. Individual transcription factor binding sites within the Egr-1 promoter were specifically mutated and Egr-1 promoter activity was tested using reporter gene constructs. Our experiments demonstrate that serum response elements are the main positive regulators and binding to a cAMP responsive element represents the major negative regulator for Egr-1 expression in synovial fibroblasts. In addition, we functionally defined a new element, which was not yet described in the human Egr-1 promoter and which serves as a second negative regulatory element for Egr-1 expression. Therefore increased serum response factor activity or failure of Egr-1 repressing signals may account for Egr-1 overexpression in RA synovial fibroblasts.