We characterized two human immunodeficiency virus type 1 (HIV-1) strains, HIVMCK and HIV213, which have different cytopathic effects in infected cells. HIV213 was highly cytopathic, whereas HIVMCK was not. Biological analyses of chimeric viruses from the cloned infectious DNAs of HIVMCK and HIV213 showed that the Vpu region was responsible for the differing cytopathicity of these viruses. Although HIVMCK expressed Vpu protein, HIV213 did not because of a mutation at the start codon for Vpu. The amounts of envelope glycoprotein and virus particles associated with the cell surface were significantly increased on cells infected with Vpu-deficient viruses compared with Vpu-positive viruses. These data suggest that the highly cytopathic effects of HIV213 (Vpu-deficient) are due to an accumulation of envelope glycoprotein at the infected-cell surface, which would be caused by the retention of progeny virions in the absence of Vpu-facilitated virion release.