Mouse cytohesin-1 (CTH-1) is a guanine nucleotide exchange factor, specific for ADP ribosylation factors. The CTH-1 gene promoter was characterized by deletion mapping and mutational analysis. The region between -101 and -38 relative to the transcription start site showed the essential promoter activity when transfected into both NIH3T3 and COS7 cells. The nucleotides of the core promoter region contain three tandem GC boxes, which can offer potential binding sites for the ubiquitous transcription factor Sp-1 family. Mutational analysis revealed that these tandem GC boxes are indispensable for activation of the gene transcription.