Treatment of flavivirus-infected mammalian and mosquito cells with acidotropic amines (such as chloroquine, ammonium chloride, or methylamine) inhibited the normal proteolytic processing of the virus prM protein to M. As a result, virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prM protein rather than M protein. Identification of the prM protein was based on molecular weight, glycosylation, and reactivity with an anti-prM monoclonal antibody. Infected cells which had not been treated with acidotropic amines did release, along with virions which contained the mature M protein, variable amounts of virus containing the prM precursor. The relative amounts of these two types of virions were influenced both by the virus and the host cell type. Virions containing the prM protein had a lower specific infectivity than virions containing the M protein; however, in experiments with a macrophage cell line this low specific infectivity was significantly increased if the anti-prM monoclonal antibody was used to facilitate virus entry via Fc receptors. Our findings indicate that the proteolytic cleavage of prM requires an acidic environment and is necessary to generate fully infectious virus. We suggest that the cleavage of prM occurs in the acidic post-Golgi vesicles.