A study was made of the effect of TRH, administered in vivo by iv infusion or added in vitro to the incubation fluid, on tissue fragments prepared from rat normotopic and ectopic pituitaries. The latter were 30-day-old pituitary grafts transplanted under the kidney capsule in hypophysectomized animals. No detectable effect of TRH was found with normotopic glands. In contrast, the neurohormone produced a large increase in GH and PRL biosynthesis in the grafts, as revealed by the rise in hormone content as well as increased incorporation of L-[3H]leucine into the two hormones. These effects of TRH 1) are dose related; 2) appear after a latent period of at least 15 min; and 3) persist, although attenuated, for some time after removing the neurohormone. In vitro release of GH and PRL by tissue fragments prelabeled with L-[3H]leucine was studied by following the appearance of the radioactive hormones in the incubation fluid. Exposure to TRH produced a prompt, 2-fold or greater increase in hormone release from grafts but not from normotopic gland fragments. The possible mechanisms whereby pituitary somatotrophs and mammotrophs removed from the influence of the central nervous system increase their responsiveness to TRH stimulation are considered.