Macrophages and T cells infected in vitro with CD4-dependent human immunodeficiency virus type 1 (HIV-1) isolates have reduced levels of CD4 protein, a phenomenon involved in retroviral interference. We have previously characterized the first CD4-independent HIV-1 X4 isolate m7NDK, which directly interacts with CXCR4 through its mutated gp120. We thus investigate CXCR4 expression in cells infected with this m7NDK CXCR4-dependent HIV-1 mutant. We present evidence of the down-regulation of CXCR4 membrane expression in CD4-positive or -negative cells chronically infected with the HIV-1 m7NDK, a phenomenon which is not observed in the CD4-dependent HIV-1 NDK parental strain. This down-regulation of CXCR4 was demonstrated by fluorescence-activated cell sorter analysis and was confirmed by the absence of CXCR4 functionality in m7NDK-infected cells, independently of the presence of CD4 protein. Furthermore, a drastic reduction of the intracellular level of CXCR4 protein was also observed. Reduced levels of CXCR4 mRNA transcripts were found in m7NDK-infected HeLa and CEM cells, reduced levels that could not be attributed to a reduced stability of CXCR4 mRNA. Down-regulation of CXCR4 on m7NDK-infected cells may thus be explained by transcriptional regulation.