We previously demonstrated that the C-nucleoside antibiotic showdomycin (1) is biologically formed from D-ribose (presumably as phosphoribosylpyrophosphate (2)) and L-glutamic acid (3) with loss of C-1 of glutamate. Here, we report the origin of the vinylic hydrogen of showdomycin: administration of [3-2H2]-L-glutamic acid (4) to Streptomyces showdoensis cultures resulted in showdomycin exhibiting a broad singlet at δ 6.79 in its 2H n.m.r. spectrum, indicating retention of hydrogen from C-3 of glutamate. Using stereospecifically labelled (2S,3S)-[3-2H]glutamic acid (5) and (2S,3R)-[2,3-2H2]glutamic acid (6), we found that (2S,3S)-labelled glutamate led to deuterium incorporation in showdomycin, while (2S,3R)-labelled glutamate did not. These results show that formation of the maleimide ring of showdomycin from L-glutamate involves retention of the 3-pro-S-hydrogen and loss of the 3-pro-R-hydrogen. The findings have implications for the mechanism of ribose-glutamate linkage and indicate that the 3-oxo intermediate theory is untenable for showdomycin biosynthesis.