The structure and activity of the pseudodisaccharide core found in aminoglycoside antibiotics was probed with a series of synthetic analogues in which the position of amino groups was varied around the glucopyranose ring. The naturally occurring structure neamine was the best in the series according to assays for in vitro RNA binding and antibiotic activity. With this result in hand, neamine was used as a common core structure for the synthesis of new antibiotics, which were evaluated for binding to models of the Escherichia coli 16S A-site ribosomal RNA, in vitro protein synthesis inhibition, and antibiotic activity. Analysis of RNA binding revealed some correlation between the relative affinity and specificity of RNA binding and antibacterial efficacy. However, the correlation was not linear. This result led us to develop the in vitro translation assay in an effort to better understand aminoglycoside-RNA interactions. A linear correlation between in vitro translation inhibition and antibiotic activity was observed. In addition, IC(50)s in the protein synthesis assay were typically lower than the K(d)s Obtained for RNA binding, suggesting that binding of these compounds to intact ribosomes is tighter in these cases than binding to the model RNA oligonucleotides. This reflects possible differences in RNA conformation between intact ribosomes and the free RNA of the model system, or possible high-affinity ribosomal binding sites in addition to the A-site RNA.