We have cloned the genomic DNA encoding the human NeuAc alpha2,3Gal beta1,3GalNAc alpha2,6-sialyltransferase (hST6GalNAc IV) and analysed its structure. The hST6GalNAc IV gene was found to span about 9 kb and to be composed of six exons. The 5'-RACE (rapid amplification of cDNA ends) results indicated that mRNA isoform of the hST6GalNAc IV was generated by alternative splicing in the 5'-untranslated region. The expression of this gene was highly restricted in human fetal tissues. The potential transcriptional start site was determined by CapSite hunting. Sequence analysis of the 5'-flanking region of this gene lacked canonical TATA and CAAT boxes, but contained several putative binding sites for transcription factors SP1, MZF1, GATA1, LMO2COM, NFAT, HFH8 and USF, etc. Functional analysis of the 5'-flanking region by transient expression method revealed a high transcriptional activity in both HepG2 cells and Molt4 cells in a cell type-dependent manner, but not in SK-N-MC cells. These results suggest cell type-specific regulation of the basal hST6GalNAc IV promoter activity.