The molecular mechanism of the phosphorylation-dependent activation of tryptophan hydroxylase is studied with respect to the role of the 14-3-3 protein. Reexamination of the system reconstituted with the purified TRH and the 14-3-3 protein showed that the level of the TRH activity correlated with the extent of the Ca2+/calmodulin- or the cAMP-dependent phosphorylation in TRH. The experiment confirmed the requirement of the 14-3-3 protein for the activation, but the 14-3-3 protein added into the assay mixture did not affect either the extent nor the specificity of the phosphorylation. However, the analysis of the assay mixture on a pteridine-based affinity column indicated the formation of a complex between TRH and the 14-3-3 protein, where the complex formation depended on the phosphorylation of TRH. The complex between the phosphorylated TRH and the 14-3-3 protein could also be detected by analysis of crude brainstem extract previously phosphorylated by endogeneous Ca2+/calmodulin-dependent protein kinase. The 14-3-3 protein, therefore, appears to be a phosphorylation-dependent TRH-binding protein whose interaction causes the activation of TRH.