Pituitary TSH (McKenzie bioassay), hypothalamic(HTH) TRF-like activity, and pituitary responsiveness to TRF were determined in fetal (18th–22nd days gestation), infant (0–23 days of age), and adult rats. Pituitary TSH concentration in-creased 10-fold between the 18th and 22nd days of gestation and, after birth, increased further to adult levels by 1 week of age. Fetal and neonatal pituitaries were capable of releasing TSH when incubated in vitro with TRF-like material extracted from the HTH of adult donor rats. HTH TRF-like activity was measured by its ability to release TSH from test mice in vivo and from donor rat pituitaries in vitro. TRF-like activity was present in the HTH of fetal rats in low concentration (20% of that in adults) and increased to adult levels by the second postnatal week. Thus, the capacity of the HTH to produce TRF and that of the pituitary to respond to TRF are present before birth and throughout the neonatal period.