The terminal step of thyroliberin (TRH) biosynthesis was studied in fetal mouse hypothalamic cultures by incorporation of labelled amino-acids. Neosynthetized labelled TRH was identified by both biochemical and immunological techniques in cell and media extracts. Three possible stimuli of TRH biosynthesis were investigated: maturation of TRH neurons in culture, acceleration of the last processing step by ascorbate supplementation of the culture medium, and K(+)-evoked release of TRH. In all three cases, rate of labelled TRH synthesis was not changed while the amount of unlabelled TRH was increased. Moreover, in the case of K(+)-evoked release of TRH, the preferential release of a preformed unlabelled pool of TRH was observed. These results suggest that regulatory mechanisms are more likely to occur at the level of the processing of stored precursor(s) rather than on their neosynthesis.