Tertiary folding of the 160-nt P4-P6 domain of the Tetrahymena group I intron RNA involves burying of substantial surface area, providing a model for the folding of other large RNA domains involved in catalysis. Stopped-flow fluorescence was used to monitor the Mg2+-induced tertiary folding of pyrene-labeled P4-P6. At 35 °C with [Mg2+] ≈ 10 mM, P4-P6 folds on the tens of milliseconds timescale with kobs = 15–31 s−1. From these values, an activation free energy ΔG[Dagger] of ∼8–16 kcal/mol is calculated, where the large range for ΔG[Dagger] arises from uncertainty in the pre-exponential factor relating kobs and ΔG[Dagger]. The folding rates of six mutant P4-P6 RNAs were measured and found to be similar to that of the wild-type RNA, in spite of significant thermodynamic destabilization or stabilization. The ratios of the kinetic and thermodynamic free energy changes Φ = ΔΔG[Dagger]/ΔΔG°′ are ≈0, implying a folding transition state in which most of the native-state tertiary contacts are not yet formed (an early folding transition state). The kobs depends on the Mg2+ concentration, and the initial slope of kobs versus [Mg2+] suggests that only ∼1 Mg2+ ion is bound in the rate-limiting folding step. This is consistent with an early folding transition state, because folded P4-P6 binds many Mg2+ ions. The observation of a substantial ΔG[Dagger] despite an early folding transition state suggests that a simple two-state folding diagram for Mg2+-induced P4-P6 folding is incomplete. Our kinetic data are some of the first to provide quantitative values for an activation barrier and location of a transition state for tertiary folding of an RNA domain.