The effect of exogenous gonadal steroids or orchidectomy on the distribution and concentration of luteinizing hormone-releasing hormone (LHRH) in the hypothalamus of the dog was studied. Various hypothalamic area of the canine brain were extracted with 90% methanol and processed, and LHRH was assayed, using a specific radioimmunoassay. The highest concentration of immunoreactive LHRH was found in the infundibular, premammillary, preoptic, and suprachiasmatic areas in intact dogs. Measurable amounts of LHRH were found in the mammillary and lateral hypothalamic regions. The hypothalamus in the male dog had significantly (P < 0.05) higher LHRH concentrations 3 mm lateral to the midsagittal line (the lateral hypothalamic area) as compared with the area in the anestrous female dog. Daily estrogen treatment of female dogs with 0.2 mg/day or 1 mg/day, IM, or with testosterone propionate (1 mg/day, IM) for 3 weeks increased LHRH concentration 2.5- to 5-fold in the mammillary, premammillary, and infundibular areas and almost 40-fold in the preoptic, rostral commissural area and in the lateral and dorsal hypothalamic regions. Orchidectomy caused an increase in LHRH concentration in the preoptic and suprachiasmatic area with a concurrent decrease in LHRH in the infundibular area. Therefore, in the dog, LHRH is possibly synthesized or transported (or both) from different regions of the hypothalamus to the median eminence, and the gonadal steroid feedback mechanism is complex, involving multiple sites of action in the hypothalamus and the components of the limbic system.