cDNA and genomic clones encoding mouse Galbeta1, 3GalNAc-specific GalNAc alpha2,6-sialyltransferase (ST6GalNAc II) were isolated, and the structure organization of the gene was determined. The predicted amino acid sequence is 57.4% identical to the chick ST6GalNAc II sequence but 33.8% identical to the chick ST6GalNA I (GalNAc alpha2, 6-sialyltransferase) sequence. The ST6GalNAc II gene is constitutively expressed in various mouse tissues but highly expressed in lactating mammary gland and adult testis. The gene contains nine exons spanning about 25 kilobases of genomic DNA and encodes a messenger RNA of 1995 nucleotides. Primer extension and S1 nuclease protection analysis of submaxillary gland mRNA showed that the transcription of the ST6GalNAc II gene starts from 68 nucleotides upstream from the translation start site. Characterization of 5'-flanking genomic regions indicated that the Galbeta1,3GalNAc-specific GalNAc alpha2,6-sialyltransferase promoter is embedded in a G+C-rich domain and contains no TATA or CAAT box but has putative binding sites for transcription factors Sp1 and AP-2. Transient transfection experiments involving luciferase reporter genes demonstrated promoter activity in NIH3T3 cells.