Human immunodeficiency virus type I (HIV-1) exploits various host cellular pathways for efficient infection. Here we report that the absence of mitochondrial DNA (mtDNA) in ρ(0) cells markedly attenuates HIV-1 infection. Importantly, reduced infection efficiency in ρ(0) cells is not simply the result of impaired oxidative phosphorylation (OXPHOS) because pharmacological OXPHOS inhibition did not inhibit HIV-1 infection. Analysis of the early steps of virus infection by real-time PCR quantification of stage-specific HIV-1 DNA products in the infected ρ(0) and parental cell line have allowed us to conclude that HIV-1 infection in ρ(0) cells is blocked at the steps that occur after reverse transcription and prior to nuclear import. Additionally, confocal fluorescence microscope analysis showed that the majority of viral complexes containing HIV-1 p24 co-localize with mitochondria in target cells, suggesting an interaction between the two. Collectively, our data strongly indicate that mitochondria play an important role during early stages of HIV-1 infection, probably through direct association with HIV-1 intracellular complexes.