Cellular DNA of the kidney from a patient with PML was analyzed by reassociation kinetics for the presence of JC virus DNA. Various amounts of viral DNA sequences were detected in different areas of the kidney. The highest concentration (175 genome equivalents/cell) was found in the renal medulla and there were almost none in the renal cortex. Differentiation from the closely related BK virus was carried out by reassociation kinetics and restriction enzyme cleavage with subsequent Southern blot analysis. The enzyme Hind II, which does not cleave within the BK virus genome, generated four restriction enzyme fragments in the cellular DNA from the kidney, thus documenting the presence of JC virus DNA. By examination of the renal DNA with the "no-cut" restriction enzyme XHO I and the "one-cut" enzymes Eco RI and BAM HI it was possible to show that free and not integrated viral DNA was present in these cells. Nonhomogeneous defective DNA bands were not detectable. By in situ hybridization the epithelial cells lining the collecting tubules were found as predominant site of the viral infection in the kidney.