In this study, quantitative assessment of the synergistic and independent effects of estradiol and progesterone on protein synthesis in the ventromedial hypothalamus (VMN) and the preoptic area (POA) was accomplished using in vitro 35S-methionine and 35S-cystein labeling, two-dimensional gel electrophoresis, and computerized densitometry. Ovariectomized (OVX) rats were divided into four groups. Group 1 was implanted with estradiol (E) capsules for 6 hr and injected with progesterone (P; 0.1 ml, 5 mg/ml propylene glycol) at 20 hr. Group 3 was sham-implanted for 6 hr and injected with 0.01 ml P at 20 hr. Group 4 was sham-planted for 6 hr and injected with vehicle alone at 20 hr. All animals were sacrificed at 24 hr. A number of proteins in both VMN and POA were found to be increased or decreased in labeling by E plus P, E alone, and P alone. Two important synergistic effects of the hormones were found. First, the effects of E on labeling of several proteins in both brain regions were countered by P, and conversely, the effects of P on labeling of several proteins in both brain regions were countered by E. Second, E priming increased the number of proteins affected in labeling by P in both brain regions. Comparison of the effects of E and P on proteins in the VMN and POA indicated that the populations of proteins affected in labeling were markedly different. These results begin to clarify the mechanism in which E and P affect neuronal functioning in two regions involved in the control of reproduction and lend support to the hypothesis that gonadal steroids accomplished their action on brain tissue via a mechanism that is partly unique to the brain region.