Human leukosialin (CD43) is a major sialoglycoprotein expressed on leukocytes and platelets. In order to investigate the transcriptional regulation of this gene, we have isolated a genomic DNA of 12 kilobases in size that includes the 5'-flanking sequence. Comparison of the genomic and cDNA sequences revealed that the leukosialin gene consists of two exons, and that its entire translation product is encoded in the second exon. The transcriptional start site was determined by primer extension analysis in human T-cell line Jurkat. No canonical TATA or CAAT boxes were found in the prospected upstream region, but a guanine-rich region was observed on the sense strand. To localize the transcriptional regulatory region of this gene, various regions of 5' sequences were fused to the chloramphenicol acetyltransferase (CAT) gene, and transient expression assays were conducted in Jurkat cells. We employed the polymerase chain reaction to generate a series of clones containing 5' sequences of the leukosialin gene using primer sequences based on the genomic sequence. This strategy enabled us to narrow the search for a transcriptional regulatory element. In addition, we introduced site-directed mutations in the regulatory region by polymerase chain reaction to define it in detail. These studies showed that the sequence from -53 to -40 base pairs 5' to the transcriptional start site is critically involved in leukosialin expression. This sequence, 5'GGGTGGGTGGAGCC3', represents a novel promoter sequence which has not been reported in the promoters for other molecules expressed in T-lymphocytes.