The nature of corticosteroid-binding globulin (CBG)-producing cells was studied in guinea pig liver by immunoperoxidase cytochemistry in light and electron microscopies. In light microscopy, CBG was detected in the cytoplasm of hepatocytes from pregnant and nonpregnant guinea pigs. The CBG-stained hepatocytes were more numerous in the peripheral regions of the lobules and around the portal space. In electron microscopy, CBG was associated with the hepatocyte rough endoplasmic reticulum cisternae. Patches of dense deposits were occasionally seen in the perinuclear cisternae and in cisternae identified as part of the Golgi apparatus. No deposits were seen in the smooth endoplasmic reticulum or any other cell organelles. Kupffer and bile duct cells proved to be negative to CBG. These findings demonstrate that the rough endoplasmic reticulum is the site of CBG synthesis in the guinea pig hepatocyte and confirm the hepatic origin of CBG, previously shown by biochemical methods. The distribution of CBG was also studied by light microscopy in other tissues from pregnant guinea pigs. No CBG was detected inside cells from muscle, heart, lung, kidney, ovary, uterus, or placenta. CBG was only detected in vascularized zones (glomeruli in the kidney, perifollicular capillary network and corpora lutea of the ovary, and connective tissue separating the myometrium layers of the uterus).