The aminoglycoside antibiotic neomycin B induces misreading of the genetic code during translation and inhibits several ribozymes. The self-splicing group I intron derived from the T4 phage thymidylate synthase (td) gene is one of these. Here we report how neomycin B binds to the intron RNA inhibiting splicing in vitro. Footprinting experiments identified two major regions of protection by neomycin B: one in the internal loop between the stems P4 and P5 and the other in the catalytic core close to the G-binding site. Mutational analyses defined the latter as the inhibitory site. Splicing inhibition is strongly dependent on pH and Mg2+ concentration, suggesting electrostatic interactions and competition with divalent metal ions. Fe2+-induced hydroxyl radical (Fe-OH.) cleavage of the RNA backbone was used to monitor neomycin-mediated changes in the proximity of the metal ions. Neomycin B protected several positions in the catalytic core from Fe-OH. cleavage, suggesting that metal ions are displaced in the presence of the antibiotic. Mutation of the bulged nucleotide in the P7 stem, a position which is strongly protected by neomycin B from Fe-OH. cleavage and which has been proposed to be involved in binding an essential metal ion, renders splicing resistant to neomycin. These results allowed the docking of neomycin to the core of the group I intron in the 3D model.