The involvement of lysosomes in infection by mouse hepatitis virus 3 (MHV3) was studied. L cells were infected with MHV3 in the presence of NH4Cl or chloroquine, weak bases which increase the intralysosomal pH and impair lysosomal functions. NH4Cl significantly inhibited virus-induced cytopathic effects and MHV3 replication, but did not prevent the attachment of 3H-labelled virus. No inhibition of MHV3 replication by NH4Cl and chloroquine was observed when lysosomotropic agents were added later than 3 h post-infection, suggesting the direct involvement of lysosomes in release of the viral genome into cytoplasm. These results, together with the lack of antibody-mediated immune lysis of MHV3-infected cells, suggest that MHV3 entered cells by an endocytic pathway (viropexis) followed by internalization into cellular lysosomes.