Progressive multifocal leukoencephalopathy (PML) is an often-fatal demyelinating disease of the CNS that usually develops in immunocompromised individuals because of reactivation of quiescent JC virus (JCV). There are only a few reports of JCV infection in the human spinal cord. Progressive multifocal leukoencephalopathy-like demyelinating lesions have been documented in the brains of simian immunodeficiency virus-infected macaques. To determine whether simian virus 40 (SV40) can infect and cause PML lesions in spinal cords of immunosuppressed macaques, we examined archival spinal cord samples from 15 simian immunodeficiency virus-infected rhesus monkeys with acquired immunodeficiency syndrome and SV40 infection of the brain. Among those, 6 (40%) had SV40-infected cells in the spinal cord, including 1 with PML-like lesions, 1 with PML-like lesions and meningoencephalitis, 2 with meningoencephalitis, 1 with gray matter gliosis, and 1 with no lesions. One animal with a large PML-like lesion had extensive demyelination and SV40 infection of astrocytes, oligodendrocytes, and meningeal cells. None of the 6 animals had SV40-infected spinal cord neurons. These observations indicate that, like JCV in immunosuppressed humans, SV40 can infect glial cells and cause PML-like lesions in the spinal cord of immunosuppressed rhesus macaques. Rhesus macaques could serve as an animal model to study polyomavirus infection and pathogenesis in the spinal cord.