The fluorescent Ub* mutant of the small globular protein ubiquitin is studied by multiple spectroscopic techniques under low temperature/high viscosity conditions to reveal unambiguously the formation of a compact structured intermediate preceding the barrier crossing to the native state. When detected by fluorescence intensity alone, ubiquitin appears to fold via a quasi two-state mechanism. In contrast, circular dichroism reveals the early formation of a structured ensemble, whose signal between 210 and 230 nm exceeds that of the native state, and whose unfolding curve is cooperative. The ensemble radius of gyration determined by small-angle X-ray scattering is only 1.2 times that of the native state, close to the classical molten globule value. Experiments performed in water/ethylene glycol mixtures at temperatures as low as -20 degreesC allow us to set an upper limit of : less than or equal to8k(B)T on the barrier height for formation of the intermediate ensemble. The very small fluorescence burst phase indicates that the tryptophan residue is not yet packed into a nativelike conformation in the intermediate ensemble and that kinetics monitored by fluorescence intensity alone are not always a reliable indicator of two-state folding.