We established 99 JC virus (JCV) DNA clones directly from the brain of a single patient with progressive multifocal leukoencephalopathy (PML). Based upon restriction patterns, the cloned viral DNAs were classified into two major groups (NY-1A and -1B) containing 53 and 35 clones, respectively, and several minor groups containing one or a few clones. The regulatory sequences of representative clones were compared with the archetypal regulatory sequence, which has been detected in JCV DNAs cloned from the urine of healthy and nonimmunosuppressed individuals. The regulatory sequence of NY-1B had the two structural features common to most PML-type regulatory regions, duplication and deletion of specific segments in the archetypal sequence, while that of NY-1A contained a small deletion and an insertion of a 29-bp sequence originating from the early region of the JCV genome. A regulatory region similar to that of NY-1A has never been detected in JCV isolates obtained thus far.