beta-Endorphin and enkephalin in extracts of whole brain, various brain regions, adenohypophysis, and combined pars intermedia and neurohypophysis of the rat were measured by radioimmunoassay. In brain extracts, the immunoreactive substances were further separated according to molecular size by gel filtration. beta-Endorphin was found in the diencephalon but not in the hippocampus, cerebral cortex, cerebellum, and striatum. Enkephalin was found predominantly in the striatum and diencephalon. Attention is called to possible artifactual interference by myelin basic protein in the immunoassays for beta-endorphin in some regions of the brain. In the pituitary, enkephalin was mainly restricted to the pars intermedia-neurohypophysis. Neither adrenalectomy nor hypophysectomy significantly altered levels of beta-endorphin in brain extracts. Adrenalectomy increased the levels of beta-endorphin in adenohypophysis and pars intermedia-neurohypophysis; after adrenalectomy, enkephalin was also increased in the adenohypophysis but less so in the pars intermedia-neurohypophysis. These results show that brain endorphin levels are independent of pituitary endorphin levels; they suggest that beta-endorphin-containing neurons and those containing enkephalin constitute two separate groups of brain cells.