The entry of African swine fever virus into Vero cells has been investigated by both biochemical and morphological techniques. A quantitative electron microscopy analysis of the early steps of the infection has shown that African swine fever virus enters Vero cells by a receptor-mediated endocytosis mechanism. The internalization of virus particles is a temperature- and energy-dependent process, since it did not take place at 4 degrees or in the presence of NaF and 2,4-dinitrophenol. To determine the involvement of acidic intracellular vacuoles in the virus entry pathway we have tested the effect of lysosomotropic agents in the infection. Chloroquine, dansylcadaverine, amantadine, methylamine, and ammonium chloride inhibited African swine fever virus production in Vero cells. Dansylcadaverine and chloroquine did not inhibit virus adsorption and internalization; however, in the presence of these drugs, virus particles were retained in cytoplasmic vacuoles and early viral RNA and protein synthesis were not detected, indicating that these compounds inhibit an early step in the infectious cycle, probably the uncoating of the virus particle.