The thermodynamics and kinetics of folding are characterized for villin 14T, a 126-residue protein domain. Equilibrium fluorescence measurements reveal that villin 14T unfolds and refolds reversibly. The folding kinetics was monitored using stopped-flow with fluorescence and quenched-flow with NMR and mass spectrometry. Unfolding occurs in a single-exponential phase in the stopped-flow experiments, and about 75% of the total amplitude is recovered in the fast phase of refolding. The remaining 25% of the amplitude probably represents trapping in cis-trans proline isomerization pathways. At 25 degreesC, the stability estimate obtained by extrapolation from the transition region of the stopped-flow chevron matches the stability value from equilibrium urea titrations (DeltaG = 9.7 kcal/mol, m value = 2.2 kcal mol-1 M-1). At low final urea concentrations, however, the refolding kinetics deviates from the two-state model, indicating the formation of an intermediate. Under these conditions, quenched-flow followed by NMR and mass spectrometry show no detectable hydrogen-bonded intermediate in the fast refolding phase. In contrast, agreement is observed between the equilibrium and kinetic estimates of stability at 37 degreesC (DeltaG = 6.0 kcal/mol, m value = 1.6 kcal mol-1 M-1), at all observed urea concentrations, demonstrating apparent two-state folding at this temperature. This result shows that the two-state folding model, previously applied to small domains with single, central hydrophobic cores, can also describe the folding of a larger domain with multiple core structures.