GM3 synthase, which transfers CMP-NeuAc with an alpha2,3-linkage to a galactose residue of lactosylceramide, plays a key role in the biosynthesis of all complex gangliosides. The expression of this gene is highly restricted in both human fetal and adult tissues. To elucidate the mechanisms that regulate the tissue-specific expression of the human GM3 synthase (hST3Gal V) gene, we have isolated and characterized its 5'-flanking region. Potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5'-flanking region revealed that hST3Gal V promoter lacked canonical TATA and CAAT boxes but contained several putative binding sites for transcription factors AP4, MZF1, SP1, ATF/CREB, NFY, IK2 and LYF1, etc. Functional analysis of the 5'-flanking region of the hST3Gal V gene by transient expression method revealed that the -177 to -83 region is important for transcriptional activity of the hST3Gal V gene in SK-N-MC and HepG2 cells. The present results also suggested that both positive and negative regulatory elements are present in this TATA-less promoter of the hST3Gal V gene.