A continuous flow incubation (perufusion) system was developed in which the secretory responses of pools of hemipituitaries from adult male rats to hypothalamic extract (HE) were characterized by serial radioimmunoassay of LH and FSH in the effluent medium. There was in initial massive release of LH and FSH which, in the absence of HE, declined to low basal levels at a rate which depended on the flow rate. Thereafter, the baseline for LH continued to decline gradually while that for FSH was stable. The rate of LH and FSH release rose abruptly after addition of the HE to the medium and returned promptly to baseline after withdrawal of the HE. During continuous infusion of HE for five hours, LH secretion was maintained at a relatively constant, elevated level. The responses to repeated identical pulses of HE were highly reproducible. The variability between responses by any one pool of tissue was significantly less than between responses of separate pituitary pools including pools comprising right and left halves of the same glands. For any given pool of pituitaries of the relationships were linear between: 1) duration of HE pulses (concentration constant) and increases in LH output, and 2) log of concentration of HE (pulse duration constant) and increases inLH and in FSH output. Consistent responses were obtained for up to 12 hr, the maximum period tested.