alpha1,3/4-Fucosyltransferase (FUT3) is involved in the synthesis of sialyl Le(a) tetrasaccharide, a tumor-associated carbohydrate antigen. Fucosyltransferases are thought to be important regulatory enzymes in the synthesis of fucosylated structures. However, there are conflicting data on the role of FUT3 in the synthesis of this carbohydrate structure and more studies on the regulation of FUT III gene expression are needed. Therefore, as first step, the promoter of FUT III gene was cloned and characterized. Sequencing data showed the absence of TATA, CAAT, and GC boxes, but many binding sites for transcription factors, previously described in colon cancer cells, were identified. Analysis of enhancer and silencing elements of deletion mutants revealed the presence of basal promoter elements of the FUT III gene in the region -636 to -674 bp from the translation initiation site, and positive and negative regulatory elements within the -674 bp to -854 bp and -854 to -1220 regions, respectively. 5'-RACE analysis showed the presence of two transcripts with 5'-ends localized within the exon A. The 5'-end of the longer transcript extended -229 nucleotides from the translation start codon and contained a sequence corresponding to an Inr element, localizing the putative transcription initiation site within this sequence. The strong correlation between the promoter activity of the FUT III gene and the high expression of sialyl Le(a) observed in different colon carcinoma cell lines seem to confirm the important regulatory role of FUT3 in the synthesis of sialyl Le(a).