Sialic acids play important roles in biological processes, such as cell-cell communication and cell-matrix interaction. Histochemical analysis using PNA and LFA lectin has shown that the expression of alpha 2,3-sialic acid linked to Gal beta 1,3GalNAc is high in basal cells and decreases following further keratinocyte differentiation. In the present study, we used an in vitro keratinocyte cell line differentiation model to study expression of alpha 2,3-sialic acid linked to Gal beta 1,3 GalNAc. Treatment of the human papillomavirus type 16-immortalized human keratinocyte (PHK16) cell line with high concentrations (1.0 mM) of Ca2+ resulted in PHK16 cell differentiation and redistribution of PNA binding glycoproteins. The synthesis of alpha 2,3-sialic acid linked to Gal beta 1,3GalNAc is mediated by three beta-galactoside alpha 2,3-sialytransferases, which are the gene products of hST30, hST30/N and hST3 Gal II. Ca2+ treatment of PHK16 cells decreased the mRNA expression of hST30/N, whereas the mRNA of hST30 and hST3Gal II was not detected by Northern blot analysis, suggesting that the hST30/N gene is responsible for sialic acid down regulation during keratinocyte differentiation. In order to examine transcriptional regulation of the hST30/N gene, we first determined the transcriptional starting sites of the hST30/N gene in PHK 16 using 5'-RACE analysis. Two kinds of type B isoforms, types B3 and BX, were identified. Type BX is a novel isoform related to the type B form, but which differs upstream of the B3 exon. The results of Northern blot analysis using a type BX-specific probe suggest that the B3 promoter may be regulated by Ca2+. Using a luciferase assay, we identified a functional DNA portion within hST30/N genomic DNA that confers negative transcriptional regulation on the hST30/N B3 promoter during Ca2+ stimulated human keratinocyte differentiation. This element contains some putative transcriptional factor binding sequence motifs such as AP2.