We have suggested in a previous study using 2-nm colloidal gold labeled-testosterone-bovine serum albumin (testosterone-BSA-gold) that 2-nm gold labeled-steroid hormone-BSA conjugates would be a useful tool for analyzing the mechanism of steroid hormone action (39). In this study, we examined whether hydrocortisone-BSA conjugate (hydrocortisone-BSA) showed a similar distribution to radiolabeled hydrocortisone in vivo, by injecting 2-nm colloidal gold labeled-hydrocortisone-BSA (hydrocortisone-BSA-gold) into the rat tail vein. The hydrocortisone-BSA-gold with silver enhancement became visible as silver deposits under electron microscopy in the nuclei of hepatocytes and hepatic stellate cells but not in Kupffer cells in the liver, and in the thymocytes and thymic reticuloepithelial cells in the thymus of a rat killed 2 h postinjection. The percentage of nuclei showing deposits in the non-target cells, the epithelial cells of the seminal vesicle, was similar to the value in the seminal vesicle of a control rat injected with BSA labeled with 2-nm colloidal gold as reported previously. In the hepatocytes and thymocytes of a control rat not injected, the percentages of nuclei showing deposits were similar to those in the rat injected with testosterone-BSA-gold or BSA-gold as reported previously, but lower than those in the rat injected with hydrocortisone-BSA-gold. These results suggest that hydrocortisone-BSA-gold is useful for the morphological study of hydrocortisone target cells, and imply that BSA conjugated with hydrocortisone can enter the target cell nuclei of the rat. The present study further indicates that the fate of gold labeled-steroid hormone-BSA conjugates may be decided at the cell membrane level.