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A substantial proportion of individuals infected with human immunodeficiency virus (HIV) also abuse alcohol. Given that each condition can disrupt brain structural integrity, with a predilection for white matter, we used MR diffusion tensor imaging (DTI) and quantitative fibre tracking to examine the separate and combined effects on the microstructure of the corpus callosum. Subjects were men and women with alcoholism alone (n=87), HIV infection alone (n=42), alcoholism and HIV infection comorbidity (n=52) and non-affected controls (n=88). The two alcoholism groups had similar lifetime alcohol consumption histories; the two HIV-infected groups had similar CD4+ counts and viral loads; all groups were matched in body mass index, and no participant was demented. Compared with controls, all patient groups had lower fractional anisotropy (FA) and higher mean diffusivity (MD) in callosal regions and fibre bundles coursing through the genu and splenium, but these effects were only significant in the two groups with alcoholism, which exhibited 0.65–1.2 SD abnormalities in FA and MD. The callosal regions were differentially affected by alcoholism, with the genu more affected than the splenium, a pattern even more pronounced in the fibre tracks. When the HIV-infected groups were divided by disease severity defined as an acquired immunodeficiency syndrome (AIDS)-defining event or low CD4+ counts (<200) and alcoholism comorbidity, the HIV-infected subgroup with AIDS and alcoholism exhibited ∼2 SD FA and MD abnormalities in the callosal sectors and fibres, abnormalities that were more than twice the effect sizes observed in the other three HIV-infected subgroups. Degradation of the callosal microstructure was consistently associated with alcoholism, with evidence for compounded alcoholism-HIV effects. Functional relevance of the microstructural abnormalities was supported by associations between motor deficits and low FA or high MD within the diagnostic groups. The high prevalence of alcoholism in HIV-infected individuals and the interfering effect of alcohol on HIV pharmacological response and therapy compliance underscore the need to recognize the independent and synergistic contributions of each condition to brain structure and function.