In a previous paper (Kim et al., 1996a), the immediate 5`-flanking region and coding region of the human UDP-N-acetylglucosamine:D-mannoside-l,4-Nacetylglucosaminyltransferase III (N-acetylglucosaminyltransferase-III; GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5`-noncoding region of the GnT III mRNA by single-strand ligation to single-stranded cDNA-PCR (5`-RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells. A cDNA clone was obtained with 5` sequences (96 bp) that diverged seven nucleotides upstream from the ATG (+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5` to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5`-noncoding region and that the cDNA was an incompletely reversetranscribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5`-flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5`-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.