Estradiol (E2) alters protein synthetic events in the ventromedial nucleus (VMN) of the hypothalamus to promote lordotic behavior in the female rat. This study analyzed the acute changes induced by E2 in proteins synthesized in the VMN in vivo as measured by 35S incorporation into protein, two-dimensional gel electrophoresis, autoradiography, and computerized optical densitometry. Ovariectomized rats received vehicle or E2, Hamilton syringes were placed stereotaxically in the VMN bilaterally, and 0.9 mCi 35S-labeled methionine and cysteine was infused over 1 h. After 6 h, rats were killed, and VMN samples were subjected to isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resultant gels underwent autoradiography, and the optical density of each of 240 spots was quantitated using a flat-bed laser scanner. Using a quantitation algorithm based on the linearity and reproducibility of the scanner and film, 123 spots were analyzed for changes in optical density and relative mol wt (MW) and isoelectric point (pI) induced by E2. The spot induced most prominently by E2 had a MW of 70K and a pI of 6.0, confirming previous results. Three spots were quantitatively induced by E2: 1) a 47K MW, pI 5.4 phosphoprotein (96% induction); 2) a 45K MW, pI 5.2 protein (72% induction); and 3) a 100K MW, pI 5.5 protein (82% induction). Two spots increased pI with E2: 1) a 110K MW protein increased from pI 5.4 to 5.5; and 2) a 50K MW protein increased from pI 6.3 to 6.4. Under these conditions and using a quantitative algorithm, only a small number of proteins synthesized in the VMN were induced by E2 in vivo. E2 also exerts effects on the posttranslational modification of another select group of proteins. These proteins may subserve at least part of the physiological effect of E2 in the VMN.