To investigate the transcriptional regulation of human GM3-synthase, a 5'-flanking fragment of 1379 bp was cloned by a PCR-based procedure. Analysis of the human genomic sequence showed that the gene consists of seven exons, locates at chromosome 2, and spans over 62 kb. There are a number of potential consensus binding sites in the cloned promoter region, but TATA and CCAAT boxes were not found in the promoter. Primer extension analysis identified two transcription start sites approximately 11 and 57 bp upstream of the exon 1. The transcription activity of the promoter was assessed in human HeLa cells by transient transfection. Of the fragments assayed, the proximal 409 bp fragment exhibits the highest transcription activity. Transcription factors that bound to the 409 bp fragment were pulled down by DNA-coupled magnetic beads. Identities of the pull-down proteins were determined by array analysis. Eight transcription factors were identified, which might either bind to the proximal region or be recruited as co-activators of the transcription factor complexes.