The three-dimensional ultrastructure of nuclei and cell organelles including rough-surfaced endoplasmic reticulum (RER), mitochondria, and cytoskeleton were studied in normal rat hepatocytes by the quick-freezing and deep-etching method. Peroxisomes and mitochondria were observed as spherical structures with granular matrices. Peroxisomes were identified by their size and matrices, which were more condensed than those of mitochondria. Ribosomes were identified as granular structures and were attached to the surface of endoplasmic reticulum. Cytoskeletal filaments were identified by their differences in diameter on the replica membranes, as well as in conventional ultrathin sections. Microfilaments were mainly localized around the bile canaliculi and adjacent to sinusoids. Intermediate filaments were observed around the bile canalicular microfilaments. Only a few filaments were observed near the lateral plasma membranes. Cross-bridges measuring 5-7 nm in diameter were localized between the lamellae of RER and the surface of mitochondria. The quick-freezing and deep-etching method could be used to clarify the three-dimensional association between the cytoskeleton and membrane-bound organelles in hepatocytes.