Although hydroxylation of tryptophan (TP) is considered to be the rate-limiting step in serotonin [5-hydroxytryptamine] synthesis, the mechanism whereby tryptophan hydroxylase (TPH) participates in the regulation of serotonin synthesis is still in question. Since the brain TP concentration is probably near the Km for tryptophan hydroxylase, changes in brain TP content could affect the rate of its hydroxylation. However, it is not established whether in vivo the activity of TPH changes with changes in TP concentration. High pressure liquid chromatography and fluorescence detection were used to measure TPH and TP in several brain nuclei and other regions of rat brain after treatments known to lower brain TP levels. Chlorimipramine, loading with neutral amino acids and a TP-deficient diet decreased the TP content of some, but not all, brain regions studied. Whenever TP content was decreased, TPH activity increased. Increased TPH activity appeared to be related to an increase in Vmax and could be prevented by blocking the TP decrease with TP injections. Several brain nuclei accumulated TP at different rates after a TP load. Possible mechanisms for regulation of serotonin synthesis in the face of fluctuating plasma levels of TP were discussed.