An organ culture system for neonatal rat anterior pituitary glands has been developed in which cellular integrity and responsiveness to LH releasing hormone (LHRH) are maintained for at least 4 days. Anterior pituitary glands from Sprague-Dawley rats were cultured individually at 37 C in an atmosphere of 95% O2-5% CO2 in BGJb culture medium, Fitton-Jackson modification. Histological examination of 5-day-old rat pituitary glands cultured for 96 h in control medium revealed no evidence of tissue necrosis. By contrast, 40-day-old-rat anterior pituitary glands were centrally necrotic after only 24 h of incubation under the same conditions. The neonatal glands were treated for 24 h with synthetic LHRH, and medium and pituitary LH FSH concentrations were determined by double antibody radioimmunoassay. On the first day of culture, LHRH caused a dose-related release of LH with the minimal effective dose between 10(-10) and 10(-9)M. Near-maximal release appeared to be attained with 10(-6)M LHRH, which induced a 12-fold elevation in medium LH over controls. Release was evident within 3 h following stimulation with 10(-9)M LHRH. Pituitary glands were responsive to this dose at 0,24 and 72 h of culture with progressively increasing ratios between LHRH-stimulated and control LH levels of 3,13, and 24, respectively. Although FSH release by LHRH was not observed on the first day of culture, on the second day LHRH caused a dose-related secretion of this hormone. Specificity of the pituitary response was demonstrated for LH and TSH release following addition of LHRH and thyrotropin releasing hormone (TRH). The simplicity, sensitivity, reproducibility, and long-term viability of this neonatal rat anterior pituitary organ culture system make it a valuable technique for the study of gonadotropin secretion.