Aminoglycoside antibiotics bind to the A-site decoding region of bacterial rRNA causing mistranslation and/or premature message termination. Aminoglycoside binding to A-site RNA decoding region constructs is established here to be only weakly stereospecific. Mirror-image prokaryotic A-site decoding region constructs were prepared in the natural D-series and the enantiomeric L-series and tested for binding to a series of aminoglycosides. In general, aminoglycosides bind to the D-series decoding region constructs with 2-3-fold higher affinities than they bind to the enantiomeric L-series. Moreover, L-neamine, the enantiomer of naturally occurring D-neamine, was prepared and shown to bind approximately 2-fold more weakly than D-neamine to the natural series decoding region construct, a result consistent with weakly stereospecific binding. The binding of naturally occurring D-neamine and its synthetic L-enantiomer was further evaluated with respect to binding to prokaryotic and eukaryotic ribosomes. Here, weak stereospecifcity was again observed with L-neamine being the more potent binder by a factor of approximately 2. However, on a functional level, unnatural L-neamine proved to inhibit in vitro translation with significantly lower potency (approximately 5-fold) than D-neamine. In addition, both L- and D-neamine are bacteriocidal toward Gram-(-) bacteria. L-Neamine inhibits the growth of E. coli and P. aeruginosa with 8- and 3-fold higher MIC than D-neamine. Interestingly, L-neamine also inhibits the growth of aminoglycoside-resistant E. coli, which expresses a kinase able to phosphorylate and detoxify aminoglycosides of the D-series. These observations suggest that mirror-image aminoglycosides may avoid certain forms of enzyme-mediated resistance.