The effect of chloroquine, a drug known to affect intracellular exocytic pathways, was studied in two retroviral systems: human immunodeficiency virus (HIV-1) and avian reticuloendotheliosis virus (REV-A). With chloroquine treatment of virus-infected cells, significant size reduction of the cell- and virus-associated surface glycoproteins, gp90 of REV-A and gp120 of HIV-1, was observed. In the case of HIV-1, extracellular virions derived from treated cells contained very little gp120. Infectivity and reverse transcriptase assays of HIV-1 demonstrated that by chloroquine treatment the majority of the virions released was noninfectious and the total virus yield was also reduced. The data suggest that chloroquine inhibition of infectious virus production is most likely due to interference with terminal glycosylation in the trans-Golgi network.