A 610-bp region of the JC virus (JCV) genome sequenced from brains of 11 progressive multifocal leukoencephalopathy (PML) patients contains 20 sites of point mutations that allow reliable classification of JCV isolates into two types. These type-determining sites were located in the region extending from position 2131 in the VP1 gene, through the intergenic region, to position 2740 in the T antigen gene. At these 20 sites the presence of different nucleotides creates two distinct patterns of substitution, with six isolates having the Type 1 pattern and five having the Type 2 pattern. Only four of the 11 isolates had 'crossovers' to the opposite type consensus DNA sequence at a small number of sites, indicating a very high type specificity. Additionally, three type-determining sites occur in the non-coding region to the left of the origin of replication. Other mutations occurred at random sites, making each strain unique, although one strain, 105, is identical to the Type 1 consensus. The JCV prototype strain Mad-1 was found to be Type 1 and differs from the consensus sequence at five sites. The other previously sequenced JCV strain, GS/B, is Type 2. At three sites out of five in the T antigen C terminus there is a type-specific amino acid substitution; however, none of the type-determining mutations in the VP1 gene cause an amino acid substitution. Comparison of each type's consensus DNA sequence to that of BK virus suggests that Type 2 represents the ancestral JCV sequence from which Type 1 diverged during human evolution.