The effects of a single stressful stimulus on serotonin (5-HT) concentration and of repeated stressful stimuli on tryptophan hydroxylase (THy) activity were measured in individual hypothalamic nuclei and other rat brain regions using specific sensitive radioisotopic enzymatic microassays. Two h after formalin injection, 5-HT levels were increased in the dorsal raphe (RDN). Acute immobilization stress, however, reduced the concentrations of 5-HT in several of the hypothalamic nuclei: the median eminence (ME), lateral amygdaloid nucleus, hippocampus, cingulate cortex (CCX) and RDN. Five consecutive daily immobilization periods failed to alter THy activity in any region examined. These results suggest a rapid release of 5-HT both from the cells and the axon terminals in the central nervous system, and failure, even with repeated stresses, to elicit induction of increased levels of the biosynthetic enzyme regulating the synthesis of the neurotransmitter.