Two mitochondrial proteins, carbamyl phosphate synthetase (CPS) and a structural membrane protein, OMM-35, were specifically localized in the hepatocyte mitochondrial matrix and inner and outer membranes, respectively, using the protein A-gold technique. Three embedding media, Epon, glycol methacrylate (GMA), and Lowicryl K4M, were tested for their ability to provide good ultrastructural preservation of mitochondrial membranes, while at the same time retaining protein antigenicity in embedded liver. Epon embedding proved to be relatively unsuitable, since mitochondrial membranes were poorly preserved. GMA and Lowicryl however gave excellent ultrastructural preservation and retained protein antigenicity sufficiently well to enable the localization of the structural membrane protein. Both qualitative and quantitative immunocytochemical demonstration of CPS have ascertained its localization to the rat hepatocyte mitochondrial matrix. The enzyme was undetectable, however, in the mitochondria of liver endothelial cells. OMM-35 was specifically located in the mitochondrial membranes and the quantitative evaluation confirms the biochemical data that OMM-35 is clearly enriched in the outer mitochondrial membrane. OMM-35 was detected in mitochondria of both hepatocytes and endothelial cells. The labeling of a relatively minor structural membrane protein such as OMM-35 gives and indication of the high sensitivity of the protein A-gold immunocytochemical technique.