Ventral prostates from adult Sprague-Dawley rats at different days postcastration were cut into one to two mm.3 pieces and incubated in medium containing S35-methionine (100 uCi/ml.) at 37C under 95% oxygen and 5% carbon dioxide for four hours. The incubated tissues were subjected to two-dimensional electrophoresis and radiofluorography. Over 100 spots were developed in the fluorograms. Three groups of spots, representing cytoskeletal proteins, androgen-dependent proteins and castration-induced proteins, were further evaluated by a computer-based densitometer. The level of densitometry absorption is proportional to the amount of radioactivity in each spot. The synthesis of cytoskeletal proteins, such as actin and tropomyosin, were relatively constant throughout the course of prostatic regression. The rate of synthesis of androgen-dependent proteins declined rapidly from a high level of synthesis before castration to a non-detectable level by Day 3 postcastration. However, three proteins, which were either not synthesized (spot G and spot H) or synthesized at a very low level (spot I) before castration, were the major proteins synthesized by the prostate during early stages of its regression. The rate of synthesis of these proteins reached a peak by Day 4 postcastration, declined rapidly and remained at a low level thereafter. The respective molecular weights and isoelectric points for these three proteins were 33 Kd and 7.2 for spot G, 38 Kd and 5.3 for spot H and 64 Kd and 6.0 for spot I. Previous findings showed that prostatic regression in rats was associated with a surge of activities in proteolytic enzymes which peaked five to six days postcastration. The peak of synthesis of three proteins noted in the present study, therefore, preceded the peak of activities of proteolytic enzymes in the regressing prostate by one to two days. Testosterone replacement to animals at the time of castration prevented the synthesis of these proteins in the prostate. Since the synthesis of these three proteins in the ventral prostate is induced by androgen-depletion resulted from castration, they are considered as the castration-induced proteins.