The synthetic D-galactose analog 2-deoxy-2-fluoro-~-galactose (dGalF) offers unique advantages for studies of the D-galactose pathway by non-invasive techniques using I9F-NMR spectroscopy or positron emission from the "F-labeled compound. The metabolism of 2-deoxy-2-fluoro-~-galactose was studied in rodents using the unlabeled, the "F-labeled, and the 14C-labeled galactose analog. Analyses for the metabolites of 2-deoxy-2-fluoro-D-galactose were performed by HPLC, enzymatic methods, and I9F-NMR spectroscopy in vivo and in vitro. The metabolism of 2-deoxy-2-fluoro-~-galactose was most active in the liver which took up the major part of the administered dose of the 14C-labeled D-galactose analog, but renal excretion was also pronounced. This was confirmed by in vivo scanning of the rat using the "F-labeled sugar (1.5 pCi/g; 25 nmol/g) and examination by positron-emission tomography and gamma camera.The dose dependence of the levels of the hepatic metabolites of 2-deoxy-2-fluoro-~-gdlactose was investigated for doses between 25 nmol/g body mass and 1 pmol/g body mass. After 1 h, the major part of the acid-soluble uracil nucleotides consisted of UDP-2-deoxy-2-fluoro-~-hexoses when the dose was at least 0.1 pmol/g. With higher doses, 2-deoxy-2-fluoro-~-galactose 1 -phosphate became the predominant initial metabolite. After a dose of 1 pmol/g 2-deoxy-2-fluoro-~-galactose 1-phosphate accumulated rapidly (5.3 0.4 kmol/g liver after 30 min) followed by the formation of UDP-2-deoxy-2-fluoro-~-galactose and UDP-2-deoxy-2-fluoro-~-glucose (0.7 50.1 pmollg and 1.8 kO.1 pmol/g, respectively, after 5 h).The diversion of uridylate, due to the accumulation of UDP-2-deoxy-2-fluoro-~-hexoses, was associated with a rapid depletion of hepatic UTP, UDP-glucose, and UDP-galactose. The UTP content was decreased to 11 5 6% of normal within 15 min after administration of 2-deoxy-2-fluoro-~-galactose at a dose of 1 pmol/g. The UTPdepleting action was minimal, however, at a dose of 25 nmol/g or less, indicating that interference in uridylate metabolism would be negligible at the doses required for positron-emission tomography of the liver using the "Flabeled compound. At higher doses, the UTP deficiency induced by 2-deoxy-2-fluoro-~-galactose could be useful in the chemotherapy of D-galactose-metabolizing tumors such as hepatocellular carcinoma. At moderate doses of this he galactose analog, I9F-NMR spectroscopy in vivo or in vitro could pinpoint defects of enzymes in galactosemia, e.g. of galactokinase, uridylyltransferase, or UDP-glucose 4-epimerase.Chemical modification of the substituents at C2 of Dgalactose results in several analogs which are metabolized via the D-galactose pathway to the respective UDP-hexose analogs [I -111. Accumulation of UDP-hexose analogs has been observed in cells and tissues metabolizing 2-deoxy-Dgalactose [l, 4, 91, 10, 111, and 2-deoxy-2-fluoro-~-galactose (dCalF) [7,8]. The meta-