Neomycin, an aminocyclitol antibiotic produced by Streptomyces fradiae, has unresolved biosynthetic questions regarding the stage at which aminoglycosyl subunits are attached and the order of their linking. Prior studies suggested paromamine (formed by glycosylating 2-deoxystreptamine with glucosamine) might be an intermediate. To clarify, we examined the incorporation of [G-14C]neamine (generated via methanolysis of [G-14C]neomycin) and [G-14C]paromamine (from [G-14C]paromomycin methanolysis) into neomycins. When [G-14C]neamine was added to 2-day-old S. fradiae cultures, it was incorporated into neomycin at 44% (with 1500-fold dilution), while lower incorporations occurred at other time points; most remaining radioactivity was in recovered neamine. An experiment confirmed neamine was incorporated without extensive degradation. In contrast, [G-14C]paromamine showed negligible incorporation into neomycin. These results support that neamine is a direct precursor for neomycin biosynthesis, whereas paromamine is not, indicating a fundamental difference from the biosynthesis of other aminocyclitol antibiotics (e.g., gentamicins, sisomicins) where paromamine-related pathways were proposed.