Metabolism of pantothenic acid (PaA) in beagle dogs was investigated. The dogs excreted 12.3% of the dose in the urine within 24 hr after a single oral administration of [3H]PaA (3 mg/kg). High performance liquid chromatographic analysis of the urine showed the presence of unchanged vitamin and a major metabolite, which accounted for 60.2 and 39.8% of the urinary radioactivity respectively. Although the metabolite was hydrolyzed by treatment with beta-glucuronidase or acid phosphatase, it was found that this hydrolysis resulted from the actions of beta-glucosidase contained as a contaminant in these enzyme preparations. beta-Glucosidase completely hydrolyzed the metabolite to generate PaA and glucose. The metabolite was isolated and subjected to GC/MS and NMR analyses. It was identical to synthetic PaA beta-glucoside, 4'-O-(beta-D-glucopyranosyl)-D-pantothenic acid. It was shown by the use of dog liver microsomes that PaA underwent beta-glucosidation in the presence of uridine diphosphate glucose (UDPG). It is proposed that beta-glucosidation by UDP-glucosyltransferase is a novel metabolic pathway of PaA in the dog.