Aminoglycoside antibiotics inhibit several types of ribozymes, including group I introns, by displacing critical Mg2+ ions. However, they stimulate activity of the small hairpin ribozyme. We show here that aminoglycosides promote self-splicing of the Cr.psbA2 group I intron at subthreshold Mg2+ concentrations. Neomycin is the most effective of the aminoglycosides tested; it stimulates splicing of Cr.psbA2 at micromolar concentrations, and, in this respect, is >100-fold more effective than spermidine. At optimal Mg2+ for Cr.psbA2 splicing, these drugs, especially kanamycin B and tobramycin, promote GTP attack at the 3' splice-site. Kinetic analysis suggests that this is due to an alternatively folded state of the ribozyme that is induced, or stabilized, by aminoglycosides. A similar effect is observed at high Mg2+ concentrations. Comparing the effects of structurally related aminoglycosides indicates that splicing promotion is more sensitive to drug structure than misfolding and occurs at lower drug concentrations. These data show that aminoglycosides can promote biochemical activities of a large ribozyme by acting as a Mg2+ mimic. The results also underscore the functional diversity of group I introns in nature.