In this study, changes in individual proteins in the ventromedial hypothalamus (VMN) and the preoptic area (POA) of the female rat brain were quantitatively assessed following either a short treatment (2 h) or a discontinuous schedule of estradiol. Ovariectomized (OVX) rats were implanted with estradiol capsules or sham-implanted for the appropriate paradigm and sacrificed by decapitation. Punches of brain tissue containing the VMN and POA were incubated with 35S-methionine and 35S-cysteine, and the labeled proteins separated by two-dimensional gel electrophoresis. Estradiol-induced changes were quantitatively assessed by computerized optical densitometry and subjected to a normalization procedure between pairs of estradiol-treated and OVX control gels. A number of proteins within the VMN and POA were found to be positively or negatively affected in labeling after either hormone administration paradigm. In both brain regions, the population of proteins affected in labeling after 2 h of estradiol treatment were markedly different from those affected after the discontinuous hormone paradigm. Comparison of the VMN and POA also indicated that the populations of proteins affected in labeling by either hormone treatment paradigm were different, with there being only 3 proteins (from a total of 39) affected in the same direction and 2 affected in the opposite direction by the hormone in both regions. These findings lend support to the hypothesis that administration of estradiol results in a molecular cascade of events within brain regions involved in the control of reproductive behavior. The differential effects of estradiol on proteins in the VMN and POA correlate with the dichotomy of function of these two brain regions and provide further evidence that gonadal steroids accomplish their action on neurons via mechanisms that are, in part, unique to the brain region.