The FK506-FKBP12 binding-domain of the kinase FRAP (FRB) forms a classic up-down four-helical bundle. The folding pathway of this protein has been investigated using a combination of equilibrium and kinetic studies. The native state of the protein is stable with respect to the unfolded state by some 7 kcal mol(-1) at pH 6.0, 10 degreesC. A kinetic analysis of unfolding and refolding rate constants as a function of chemical denaturant concentration suggests that an intermediate state may be populated during folding at low concentrations of denaturant. The presence of this intermediate State is confirmed by refolding experiments performed in the presence of the hydrophobic dye 8-anilinonaphthalene-1 sulfonate (ANS). ANS binds to the partially folded intermediate state populated during the folding of FRB and undergoes a large change in fluorescence that can be detected using stopped-flow techniques. Analysis of the kinetic data suggests that the intermediate state is compact and it may even be a misfolded species that has to partially unfold before it can reach the transition state. Folding and unfolding rate constants in water are approximately 150-200 s(-1) and 0.005-0.06 s(-1), respectively, at neutral pH and 10 degreesC. The folding of FRB is somewhat slower than for other all-helical proteins, probably as a consequence of the formation of a metastable intermediate state. The folding rate constant in the absence of any populated intermediate can be estimated to be 8800 s(-1). Despite the presence of an intermediate state, which effectively slows folding, the protein still folds rapidly with a half-life of 5 ms at 10 degreesC. The dependence of the rate constants on denaturant concentration indicates that the transition state for folding is compact with some 80% of the surface area exposed in the unfolded state buried in the transition state. Data presented for FRB is compared with kinetic data obtained for other all-helical proteins.