It has been previously shown that some toxic effects of high concentrations of glucose are mediated by the hexosamine biosynthesis pathway and its rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). We have used the cloned human GFA cDNA to study the chromosomal localization of the gene and tissue distribution of mRNA. The human GFA gene is on chromosome 2, band p13 as determined by fluorescence in situ hybridization. An 8-kb species of GFA mRNA was detected in all rat tissues tested with relatively high expression in testis and smooth muscle; a unique 3-kb mRNA species was found only in testis.