Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor beta receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.