A protein encoded by a new gene with approximately 75% homology to glutamine-fructose-6-phosphate amidotransferase (GFAT) was termed GFAT2 on the basis of this similarity. The mouse GFAT2 cDNA was cloned, and the protein was expressed with either an N-terminal glutathione S-transferase or His tag. The purified protein expressed in mammalian cells had GFAT activity. The K m values for the two substrates of reaction, fructose 6-phosphate and glutamine, were determined to be 0.8 mM for fructose 6-phosphate and 1.2 mM for glutamine, which are within the ranges determined for GFAT1. The protein sequence around the serine 202 of GFAT2 was conserved to the serine 205 of GFAT1, whereas the serine at 235 in GFAT1 was not present in GFAT2. Previously we showed that phosphorylation of serine 205 in GFAT1 by the catalytic subunit of cAMP-dependent protein kinase (PKA) inhibits its activity. Like GFAT1, GFAT2 was phosphorylated by PKA, but GFAT2 activity increased approximately 2.2-fold by this modification. When serine 202 of GFAT2 was mutated to an alanine, the enzyme not only became resistant to phosphorylation, but also the increase in activity in response to PKA also was blocked. These results indicated that the phosphorylation of serine 202 was necessary and sufficient for these alterations by PKA. GFAT2 was modestly inhibited (15%) by UDP-GlcNAc but not through detectable O-glycosylation. GFAT2 is, therefore, an isoenzyme of GFAT1, but its regulation by cAMP is the opposite, allowing differential regulation of the hexosamine pathway in specialized tissues.