To elucidate the mechanism underlying the regulation of human GD3 synthase gene expression in human melanoma SK-MEL-2 cells, we identified the promoter region of the human GD3 synthase gene. The 5'-rapid amplification of cDNA end (5'-RACE) using mRNA prepared from SK-MEL-2 cells revealed the presence of multiple transcription start sites of human GD3 synthase gene. Promoter analyses of the 5'-flanking region of the human GD3 synthase gene using luciferase gene reporter system showed the strong promoter activity in SK-MEL-2 cells. Deletion study revealed that the region as the core promoter from -1146 to -646 (A of the translational start ATG as position +1) was indispensable for endogenous expression of human GD3 synthase gene. This region lacks apparent TATA and CAAT boxes but contains putative binding sites for transcription factors c-Ets-1, CREB, AP-1 and NF-kappaB. Electrophoretic mobility shift assays using specific competitors, chromatin immunoprecipitation assay and site-directed mutagenesis demonstrated that only NF-kappaB element in this region is required for the promoter activity in SK-MEL-2 cells. These results indicate that NF-kappaB plays an essential role in the transcriptional activity of human GD3 synthase gene essential for GD3 synthesis in SK-MEL-2 cells.