Apomyolgobin (apoMb) is an important model for understanding the folding mechanism of helical proteins. This study focuses on a partially structured state of sperm whale apoMb populated at pH 4.2 (M-state), which structurally resembles a late kinetic intermediate in the formation of the native state (N) at higher pH. The thermodynamics and cooperativity of apoMb folding at pH 4.2 and 6.2 were studied by global analysis of the urea-induced unfolding transitions monitored by tryptophan fluorescence and circular dichroism. The kinetics of folding and unfolding of apoMb at pH 4.2 was measured over a time window from 40 to 850 μs, using fluorescence-detected continuous-flow measurements. Our observation of biphasic kinetics provides clear evidence for rapid (<100 μs) accumulation of previously unresolved intermediate states in both refolding and unfolding experiments. Quantitative kinetic modeling of the results, using a four-state mechanism with two intermediates on a direct route between the unfolded and folded states (U↔I↔L↔M), gave new insight into the conformational states and barriers that precede the rate-limiting step in the formation of the N-state of apoMb.