Tissue-specific carbohydrate structures are thought to result from the selective expression of specific terminal glycosyltransferases responsible for their synthesis. However, little is known about the regulation of the expression of these enzymes. Previous analysis of the distribution of one such enzyme, beta-galactoside alpha 2,6-sialyltransferase, revealed that its expression is tissue restricted, with highest levels being found in the liver. Examination of the gene suggested that its expression is regulated at the level of transcription by multiple promoters, one of which is strongly active in the liver. In this present work, an analysis of the liver-restricted promoter was undertaken to identify the promoter elements necessary for liver-restricted expression. Footprinting studies, 5' deletion analysis, and site-directed mutagenesis identified two cis-elements which were potentially important in the tissue-specific expression of this promoter. One of these elements contains a consensus binding site for the liver-enriched transcription factor hepatocyte nuclear factor-1 alpha, while the other is a consensus binding site for the liver-specific factors D-binding protein and liver-enriched transcriptional activator protein. Expression vectors containing cDNAs of these factors are capable of trans-activating transcription of the alpha 2,6ST promoter, demonstrating their ability to regulate transcription of this promoter. Together, these results suggest that tissue-specific glycosylation can be regulated at the level of transcription by the same factors involved in the expression of a number of other tissue-specific genes.