Non-denatured clathrin from coated vesicles of porcine brain was prepared in the form of reconstituted (after solubilization in buffer containing 2 M urea) polygonal coat structures. Guinea pig antibodies against this protein were characterized by immunoreplica and immunoprecipitation techniques and showed cross-reaction between clathrin from higher mammals (pig, cow, man) but not with clathrin of rat, mouse, rat kangaroo, chicken, and amphibia. Such immunologically detected interspecies differences seemed to be only minor aspects since no differences were found in proteolytic cleavage pattern analyses of clathrin from different species and tissues. Immunofluorescence microscopy using these antibodies on a variety of cells grown in culture (human embryonic brain cells, WI38 fibroblasts, HeLa cells; bovine MDBK cells and lens-forming cells) showed a typical punctate staining pattern dispersed over most of the cytoplasm but also revealed an extended juxtanuclear reticulate membrane structure. Electron microscopic immunolocalization using peroxidase techniques indicated that the antibodies preferentially, if not exclusively, reacted with clathrin present in distinct coated pits and vesicles (“dots”) and in more extended peridictyosomal membrane elements (“the reticulate structure”). On frozen sections through bovine tissues (e.g., bovine mammary gland, small intestine and liver) and human liver antibody staining was especially intense in apical and lateral regions of epithelial cells. Punctate patterns of immunostaining, indicative of localization to individual coated vesicles and pits, were also observed on tissue sections and were especially prominent in certain cell types, such as liver sinusoidal cells of the reticulo-endothelial system. The results confirm that clathrin is a protein markedly conserved during evolution but they also show that some immunologically detectable differences exist between clathrin from different species. Immunolocalization demonstrates that clathrin ( i) is enriched on distinct coated pits and vesicles, which in cultured cells usually appear to be widely dispersed throughout the cytoplasm; and ( ii) is also conspicuously associated with a certain type of juxtanuclear membrane structures. The content and the distribution of clathrin-containing structures seems to be greatly different in different types of cells and may be related to the frequency of membrane translocation processes involving clathrin coats in the specific cells and cell regions.