The presence of a number of minor components was demonstrated in commercial samples of neomycin by column chromatography on a weakly acidic ion exchange resin (Amberlite CG-50). Some of these components were isolated by ion exclusion chromatography and identified. The identification was based on chromatographic and nmr data and on mass spectrometry of volatile derivatives. The sequence of elution from the Amberlite column was : paromamine, mono-N-acetyl neamine (LP-A), paromomycin II, a diamino-dideoxyhexosyl-myo-inositol (component G), paromomycin I, mono-N-acetylneomycin C (LP-C), neamine, mono-N-acetylneomycin B (LP-B), neomycin C and neomycin B. The LP-B fraction also contained a component (K) whose gross structure corresponds to a neomycin, in which the neosamine C linked to the deoxystreptamine subunit is missing. The presence of the components LP-A, G and K has not previously been reported.