The higher order folding process of the catalytic RNA derived from the self-splicing intron of Tetrahymena thermophila was monitored with the use of Fe(II)-EDTA-induced free radical chemistry. The overall tertiary structure of the RNA molecule forms cooperatively with the uptake of at least three magnesium ions. Local folding transitions display different metal ion dependencies, suggesting that the RNA tertiary structure assembles through a specific folding intermediate before the catalytic core is formed. Enzymatic activity, assayed with an RNA substrate that is complementary to the catalytic RNA active site, coincides with the cooperative structural transition. The higher order RNA foldings produced by Mg(II), Ca(II), and Sr(II) are similar; however, only the Mg(II)-stabilized RNA is catalytically active. Thus, these results directly demonstrate that divalent metal ions participate in general folding of the ribozyme tertiary structure, and further indicate a more specific involvement of Mg(II) in catalysis.