To study the biosynthesis of the pseudotrisaccharide antibiotic, validamycin A (1), a number of potential precursors of the antibiotic were synthesized in H-2-, H-3-, Or C-13-labeled form and fed to cultures of Streptomyces hygroscopicus var, limoneus. The resulting validamycin A from each of these feeding experiments was isolated, purified and analyzed by liquid scintillation counting, H-2- or C-13 NMR Or selective;ion monitoring mass spectrometry (SIM-MS) techniques. The results demonstrate that 2-epi-5-epi-valiolone (9) is specifically incorporated into 1 and labels both cyclitol moieties. This suggests that 9 is the initial cyclization product generated from an open-chain C-7 precursor, D-sedoheptulose 7-phosphate (5), by a DHQ synthase-like cyclization mechanism. A more proximate precursor of 1 is valienone (11), which is also incorporated into both cyclitol moieties. The conversion of 9 into 11 involves first epimerization to 5-epi-valiolone (10), which is efficiently incorporated into 1, followed by dehydration, although a low level of incorporation of Zepi-valienone (15) is also observed. Reduction of 11 affords validone (12), which is also incorporated specifically into, but labels only the reduced cyclitol moiety. The mode of introduction of the nitrogen atom linking the two pseudosaccharide moieties is not clear yet. 7-Tritiated valiolamine (8), valienamine (2), and validamine (3) were all not incorporated into 1, although each of these amines has been isolated from the fermentation, with 3 being most prevalent. Demonstration of in vivo formation of [7-H-3]validamine ([7-H-3]-3) from [7-H-3]-12 suggests that 3 may be a pathway intermediate and that the nonincorporation of [7-H-3]-3 into 1 is due to a lack of cellular uptake. We thus propose that 3, formed by amination of 12, and 11 condense to form a Schiff base, which is reduced to the pseudodisaccharide unit, validoxylamine A (13). Transfer of a D-glucose unit to the 4'-position of 13 then completes the biosynthesis of 1. Other possibilities for the mechanism of formation of the nitrogen bridge between the two pseudosaccharide units are also discussed.