A mutant of Streptomyces clavuligerus (dcl-8) blocked in clavulanic acid production was found to accumulate three N-acyl derivatives of the β-lactam clavaminic acid in the culture broth. These metabolites were isolated via imidazole derivatization, reverse-phase high-performance liquid chromatography (HPLC), and aqueous column chromatography. Their structures (3, 4, 7, 8) were elucidated using 1H/13C nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy, mass spectrometry, and synthetic verification—sodium 9-aminodeoxyclavulanate (prepared from clavulanic acid) was reacted with acetylglycine to synthesize (6), which was identical to natural metabolite (3) by NMR and HPLC but had opposite CD spectra, confirming they are enantiomers. CD spectra indicated all natural metabolites possess the 3S,5S stereochemistry. Metabolite (3) exhibited negligible β-lactamase inhibitory activity, while its 3R,5R enantiomer (6) was a potent inhibitor. We conclude that S. clavuligerus dcl-8 is blocked in the clavulanic acid biosynthetic pathway between clavaminic acid and clavulanic acid, resulting in intracellular clavaminic acid accumulation that is subsequently acylated and excreted into the external medium.