Although numerous reports have appeared showing high levels of galactitol in the urine of patients with galactose-1-phosphate uridylyltransferase deficiency, little attention has been paid to measurement of urinary galactonate. Herein we explored the use of1H and13C nuclear magnetic resonance, which required only the concentration of urine without derivatization, to detect and quantitate urinary galactonate. We report that transferase deficient infants, as well as adults on galactose restricted diets excrete significant amounts of galactonate, whereas none is detected in the urine of normal subjects. Galactose-toxic infants were found to excrete large amounts of galactonate, which decreased when the lactose-free diet was instituted. We also found that normal individuals subjected to an oral galactose load also excrete high levels of galactonate for at least 4 h after galactose ingestion. Our data provide evidence that the first reaction in the oxidative pathway of galactose metabolism described in rat liver in 1966 is activated in patients with a variety of galactose-1-phosphate uridylyltransferase gene mutations even while on a lactose-restricted diet. In both patients and normal individuals, flux through the alternate galactonate pathway appears to be related to the body galactose burden.