A study was made to compare the LH and FSH release patterns from isolated adult rat pituitaries in response to exposure to acidic extracts of rat hypothalamus or brain or to acidic extracts of GnRH-depleted hypothalamus, cerebral cortex, or cerebellum of adult (90-100 days old) or young (14 days old) rats of both sexes, using a continuous perifusion system. Hypothalami of adult rats contained 3-5 ng of radioimmunoreactive GnRH, while the corresponding cerebral cortex and cerebellum contained none, or extremely low levels of GnRH. Adult hypothalamic extract (HE) stimulated considerably greater LH and FSH release than was induced by cortical extract (CE) or cerebellar extract (CBE). Removal of assayable GnRH from HE by incubation with anti-GnRH serum reduced, but did not eliminate, release of both LH and FSH, suggesting that GnRH is the principal but not sole agent responsible for both LH and FSH secretions. Adult male HE released slightly more LH and FSH than did female HE due to its greater GnRH content. GnRH-depleted CE or CBE from both sexes induced LH and FSH releases qualitatively and quantitatively similar to those induced by CE, CBE or GnRH-depleted HE. Untreated extracts or GnRH-depleted extracts of all three brain regions from 14-day-old rats of both sexes induced similar LH and FSH releases as those of adult CE or CBE. Hypothalamus of young rats, which contained about 0.5 ng of immunoassayable GnRH did not release more LH and FSH than the corresponding cortex or cerebellum did. The data indicate that all brain regions so far studied, regardless of sex or age, contained presumably nonspecific substance(s), other than GnRH, capable of stimulating minor but significant LH and FSH releases. Their nature or role in physiological regulation of gonadotropin secretion is unknown, but must be considered, since current concepts, in which GnRH is the sole hypothalamic gonadotropin regulator, are not adequate.