D-[6-3H3]6-Deoxy-5-ketoglucose(10) and D-[5,6-3H2]6-deoxyglucose (ll) were incorporated into neomycins B and C using a growing culture of Streptomyces fradiae. D-[6-3H]6-Deoxy-5-ketoglucose was incorporated into neomycin, as efficiently as the well established precursor D-glucose, and wasfound to label exclusively the 2-deoxystreptamine ring of the antibiotic. The results strengthened the previous proposals that in the formation of 2-deoxystreptamine the C-6 hydroxyl group of D-glucose is removed prior to the cyclisation reaction. Studies using the incorporation of d-[3-3H]glucose, D-[3,4-3H2]glucose and D-[5-3H]glucose into neomycin followed by the degradation of the latter established that in the biosynthesis of the 2-deoxystreptamine ring the C-4 and C-5 hydrogen atoms of glucose are removed. The loss of the C-4 hydrogen atom of the glucose is attributed to the formation of a 4-keto derivative which facilitates the removal of the C-5 hydrogen atom thus setting the stage for the expulsion of the C-6 hydroxyl group. The 5,6-olefinic intermediate formed in the process then undergoes cyclisation eventually releasing 2-deoxyinosose. The enzyme systems which participate in the conversion of D-glucose equivalent into 2-deoxyinosose may be described as 2-deoxyinosose synthase that in broad mechanistic terms resembles dehydroquinate synthase.A large group of clinically useful antibiotics, to which streptomycin and neomycin belong, are classified as aminoglycoside-aminocyclitol antibiotics. The name highlights the fact that these antibiotics, in addition to an array of variously modified amino-sugars, contain an unusual cyclohexane derivative which can either be hexa-substituted as the streptamine unit (1) of streptomycin or penta-substituted as the 2-deoxystreptamine ring (2). The latter unit is present in neomycin (3) and more than fifty other related antibiotics. It is now knownthat the carbon skeletons of both types of aminocyclitols (1 and 2) arise from D-glucose1>2) and the elegant studies of Walker and Walker3) have established that, in the formation of the streptamine ring system of streptomycin, the key event is the inositol synthase catalysed cyclisation of glucose 6-phosphate into inositol 1-phosphate, Scheme 2. The latter, through a multistage process, is then converted into streptidine (this is a diguanidinated derivative of 1). The knowledge of the latter biosynthesis provided the stimulus to consider the possibility that a cyclisation process similar to that shown in Scheme 2, followed by the reductive removal of the unwanted hydroxyl group, may also be involved in the formation of 2-deoxystreptamine. Gradually however observations were made which questioned this hypothesis and suggested that the two aminocyclitols (1 and 2) may be synthesised by two entirely different mechanisms. For example, the patterns for the incorporation of the C-skeleton ofD-glucose f Present address: