Spontaneous and induced TRH release from fragments of the male rat mediobasal hypothalamus (MBH) superfused in vitro for several h were studied by means of a specific and sensitive RIA. Recovery of exogenous TRH added to superfusion chambers with or without tissues was 98%. The spontaneous release of TRH over the initial 60 min of superfusion was 0.31 pg/MBH · min. The MBH TRH content was 2128 pg/MBH or 238 pg/mg after 4 h of superfusion. Results from serial dilutions of MBH superfusates and MBH extracts showed inhibition curves parallel to that of synthetic TRH. In a chromatographic column system, similar samples eluted undistinguishable from synthetic TRH. A high dose of K+ given as a pulse triggered an immediate TRH release (280% of basal) that peaked during the first 10 min of collection. MBHs stimulated with a 1-h 60-mM K+ infusion sustained a 7-fold increase in TRH release. Pulses of serotonin (initial concentration in chambers, 2 × 10-8 M) stimulated TRH release to 466% of basal control values. Mor...