Until just a few years ago, kinetic studies of protein folding focused primarily on processes that occur on time-scales of milliseconds or longer—a limitation set by stopped-flow instrumentation. A number of factors motivated efforts to develop new experimental methods that could be used to characterize the kinetics of more rapid processes. First, a large body of theoretical work suggested that folding on the stopped-flow time-scale usually resulted from the escape of misfolded or partially folded structures from traps in the energy landscape. Folding by direct routes from the unfolded to the native state was suggested to occur much more rapidly (1–6). Second, the observation of significant unresolved amplitude in the fluorescence and circular dichroism signals in stopped-flow experiments suggested that significant structural organization could take place in the dead time of these experiments (7-12). Rapid formation of both native-like secondary (7–11) and tertiary (8–10,12) structure was observed within the stopped-flow dead time. Data on the dynamics of secondary structure formation for synthetic α-helical peptides (13,14) and on the rate of chain diffusion under denaturing conditions (15–18), which suggested that helices and loops could form within microseconds, provided additional evidence that many interesting folding events occur on the submillisecond time scale.