JC virus, an agent that causes a human demyelinating disease, progressive multifocal leukoencephalopathy (PML), is known to induce brain tumors in hamsters. The regulatory region of the virus reported to be implicated in its tissue specificity and to be highly variable among different isolates. In a study of the regulatory region, first we isolated the JC virus genome directly from the brain of a patient with suspected PML. We amplified the regulatory region of the virus by using the polymerase chain reaction, and we compared with three JC viruses which had been isolated previously (MAD-1, MAD-2, and TKY-1). The brain tissue was shown to be infected with JC virus, as confirmed by the presence of viral antigen on immunohistochemical testing and by the demonstration of viral nucleic acids upon in situ hybridization. Southern blot analysis of the regulatory region disclosed that the isolate from our patient, which was designated as NYS-1, was roughly homologous in all three types of JC viruses, with preservation of the constant regions. However, the cut site for PvuII in NYS-1 was different from that in MAD-1 and MAD-2, and a pair of cut sites, for Nco I-PvuII, was unique to TKY-1 and absent in NYS-1; this showed that NYS-1 was a new type of JC virus. Second, we compared the regulatory regions of TKY-1 and its viral DNA integrated in a hamster brain tumor cell line (I-23) induced by TKY-1. We found no great difference between these two genomes.(ABSTRACT TRUNCATED AT 250 WORDS)