Simian virus 40 (SV40) disease was diagnosed in four rhesus monkeys that died with SIV-induced acquired immunodeficiency syndrome (AIDS). One juvenile monkey seroconverted for SV40 6 months after inoculation with SIV and developed severe bilateral tubulointerstitial nephritis. In contrast, progressive multifocal leukoencephalopathy (PML) occurred in two adult monkeys that were seropositive for SV40 before SIV inoculation, as well as a third adult that was naturally infected with SIV and seropositive for SV40 5 years before death. Large intranuclear inclusions containing abundant polyomavirus particles were limited to either renal tubular epithelial cells or oligodendrocytes. In situ DNA hybridization for SV40 large T antigen further demonstrated that SV40 nucleic acid was localized to either kidney or brain tissue. By immunohistochemical analysis, areas of central nervous system inflammation and demyelination were shown to contain CD68+ macrophages (gitter cells), aggregates of CD8+ T lymphocytes, and numerous gemistocytic astrocytes that labeled for glial fibrillary acidic protein. These observations indicate that rhesus monkeys with SIV-induced AIDS are predisposed to polyomaviral disease, in which SV40 nucleic acid is observed in renal tissue in primary infections and brain tissue after viral reactivation. Furthermore, this organ-specific replication suggests that tissue-tropic strains of SV40 may develop in immunodeficient monkeys.