Our objective was to investigate the role of the human polyomavirus JC virus as a possible cause of renal damage in AIDS subjects. Histology, immunohistochemistry, and molecular biology were used to evaluate the frequency of viral infection, genotypes, viral status, and the presence of rearrangements or point mutations in specific genomic regions of strains isolated from renal tissue. Formalin-fixed, paraffin-embedded sections of postmortem renal specimens obtained from 111 unselected AIDS patients were stained for routine histology and with anti-SV40 antibody. The immunohistochemically positive specimens were further investigated by means of nested polymerase chain reaction for different polyomavirus genomic regions (large T, transcriptional control region, and viral protein 1). Furthermore, the sequences of transcriptional control region and viral protein 1 were also analyzed. Immunohistochemistry was positive in seven cases (6.3%), four of which showed morphological evidence of viral replication (intranuclear inclusion bodies and/or intratubular cellular casts): in all seven cases, only epithelial tubular cells (with and without inclusion bodies) and cellular casts were stained. The JC virus genome was identified by polymerase chain reaction in five of the seven immunohistochemically positive cases; transcriptional control region and viral protein 1 were amplified in, respectively, three and four cases. Transcriptional control region sequence analysis revealed major rearrangements in all three cases, with duplications of all the transcriptional factor-binding sites, whereas no point mutations were found in the viral protein 1 region, which was characterized as Type 1A in all cases. For the first time in AIDS subjects, this study shows that although rarely, JC virus can replicate in renal tissue. Molecular biology revealed major rearrangements in the transcriptional control region that, together with other unknown factors, could justify the increased pathogenicity of this human polyomavirus.