Hepatitis A virus (HAV) replication in BS-C-1 cells was studied in the presence of ten potential uncoating inhibitors. Strong inhibition of HAV replication was only observed in the presence of the phenothiazine compound chlorpromazine and the lysosomotropic agent chloroquine, but not by other lysosomotropic agents. Chlorpromazine and chloroquine were found to prevent virus uncoating. Chlorpromazine is known to inhibit endocytosis of non- clathrin-coated vesicles. Chloroquine is a weak base amine, and thought to inhibit virus replication by preventing endosomal acidification. These results therefore suggest that entry of HAV in BS-C-1 cells does not depend on the low pH encountered in the clathrin-coated endocytic entry pathway. A possible role of calcium ions in mediating viral uncoating is discussed, as calcium ions were found to destabilize HAV particles in vitro.