Biosynthetic origin of 2H-tetrahydro-4,6-dioxo-1,2-oxazine, an unprecedented structural unit first discovered in alchivemycin A (1), was investigated by feeding (13)C-labeled precursors. Incorporations of both [1-(13)C]glycine and [1-(13)C]-N-hydroxyglycine into the carbon at the 4-position of this six-membered ring indicate that the hydrooxazine ring is assembled through a PKS-NRPS hybrid pathway. Additionally, alchivemycin B (2), a deoxygenated analog of 1, was isolated and its relative and absolute configurations were determined by spectroscopic analysis including NMR and CD and X-ray crystallography.