In order to determine the involvement of cholinergic transmission in the hypothalamic control of gonadotropin secretion, the activity of AChE and ChAc, enzymes taken as indices of ACh metabolism, and total protein content were measured in the hypothalamus of male and female rats under a number of endocrinological conditions. The inductive effect of testosterone on the functional differentiation of the hypothalamus immediately after birth was also investigated and the data from both experiments were correlated. The results obtained indicate that the synthesis and hydrolysis of ACh in the hypothalamus differ between males and females, from puberty to adulthood, and within the estrous cycle of the female, and that these differences are selectively localized in the preoptic suprachiasmatic area of the hypothalamus. In addition, the reduction in enzymatic activity in the postpuberal female injected neonatally with testosterone suggests that the development of this neurotransmitter system is dependent on the early hormonal environment. All of these observations imply a close parallelism between the effects of sex steroids on biochemical (neurotransmission) and functional (gonadotropin secretion) maturation of the hypothalamus.