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A significant decrease in the specific activity of 3 enzymes of serine and one-carbon metabolism (3-phosphoglycerate dehydrgenase, phosphoserine phosphatase, and serine hydroxy-methyltransferase) was found in the rat prostate gland with castration. A single injection of testosterone propionate to rats 3 days after castration resulted in a significant increase in the 3 enzyme activities within 24 h. This increase in specific activity was maximal 72 h after injection of testosterone in the case of 3-phosphoglycerate dehydrogenase and serine hydroxymethyltransferase. When cycloheximide was administered in conjunction with testosterone, the increase in 3-phosphoglycerate dehydrogenase and serine hydroxy-methyltransferase activity was significantly reduced compared to injection of testosterone alone. N6, O2'-dibutyryl adenosine-3',5'-cyclic monophosphate [(Bu)2 cAMP] and theophylline injected at 8 h intervals to rats 3 days after castration failed to mimic the action of testosterone on these 3 enzymes 24 and 72 h after beginning injections. Castration had no effect on the specific activity of these enzymes in the kidney; however, 3-phosphoglycerate dehydrogenase was significantly diminished in the liver 6 days after castration. A single injection of testosterone to rats 3 days after castration restored the activity to sham-operated levels. Serine hydroxy-methyltransferase and phosphoserine phosphatase activity in the liver were unaffected 6 days after castration. Thus testosterone exerts a regulatory role on serine and one-carbon metabolism in the prostate and liver which (Bu), cAMP is unable to minic.