Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus NRRL 2388, offers a distinct carbon skeleton structure for development of antibacterial agents targeting the bacterial ribosomal peptidyl transferase. A 31.5 kb genomic DNA region covering the hygromycin A biosynthetic gene cluster has been identified, cloned, and sequenced. The hygromycin gene cluster has 29 ORFs which can be assigned to hygromycin A resistance as well as regulation and biosynthesis of the three key moieties of hygromycin A (5-dehydro-α-L-fucofuranose, (E)-3-(3,4-dihydroxyphenyl)-2-methylacrylic acid, and 2L-2-amino-2-deoxy-4,5-O-methylene-neo-inositol. The predicted Hyg26 protein has sequence homology to short-chain alcohol dehydrogenases and is assigned to the final step in production of the 5-dehydro-α-L-fucofuranose, catalyzing the reduction of α-L-fucofuranose. A hyg26 mutant strain was generated and shown to produce no hygromycin A but 5″-dihydrohygromycin A, 5″-dihydromethoxyhygromycin A, and a 5″-dihydrohygromycin A product lacking the aminocyclitol moiety. To the best of our knowledge, these shunt metabolites of biosynthetic pathway intermediates have not previously been identified. They provide insight into the ordering of the multiple unusual steps which compromise the convergent hygromycin A biosynthetic pathway.