A biochemical and morphological investigation of the mechanism of entry of vesicular stomatitis virus (VSV) into host cells of mammalian (HeLa), avian (CER), piscine (EPC) and arthropod (Aedes albopictus) origin, is described. VSV was capable of infecting all cell lines tested by a endosome- and/or a lysosome-dependent step since ammonium chloride and amantadine blocked the early stages of infection. Complement-dependent immune lysis of infected host cells provided evidence that in none of the four different cell types examined did insertion of VSV antigens occur from the outside to any great extent on the cell surface. When the entry process was studied by electron microscopy, virus particles were seen to be bound to the cell surface at 0 degrees C. After warming at 37 degrees C for homeothermic cells or at 26 degrees C for poikilothermic cells, virus was detected within coated pits and coated vesicles and, later, in lysosomes. VSV entry was seen to take place by endocytosis in all four cell lines, which were derived from phylogenetically unrelated species.