Time-resolved fluorescence intensity and anisotropy decay measurements have been carried out on barstar, the inhibitor protein of the bacterial ribonuclease, barnase. The intrinsic fluorescence of the three tryptophans in this protein have been used to characterize the molten globule-like conformation at pH 3 (A form) and the native conformation at pH 7 (N state). The fluorescence intensity decay could be fitted to a sum of two exponentials with lifetimes of 4.1 and 1.5 ns at pH 7 (N state) and three exponentials with lifetimes of 4.9, 1.5, and 0.2 ns Bt pH 3 (A form). The emergence of the 0.2-ns component was pH dependent with a pK of similar to 4.5. Fluorescence quenching by iodide has shown that the tryptophan (Trp) residues are solvent inaccessible at pH 7 and partially exposed at pH 3 (A form). Quenching by acrylamide has suggested that the 1.5-ns decay component arises from one of the three Trp residues and the 4.1-ns component arises from the remaining two Trp residues. Of the latter, one is buried and the other is highly accessible to acrylamide. Decay of fluorescence anisotropy has shown that the Trp residues are rigidly held and do not have any segmental mobility at pH 7. The A form is characterized by a high level of aggregation and a high degree of internal motion. The aggregated A form could be relevant in the folding pathway of barstar when the possibility of interaction of molten globular form with chaperone proteins is recognized. Comparison of the dynamic behavior of the Cys --> Ala mutant with that of the wild type ha’s shown the proximity of SH group(s) to Trp residues.