JC virus (JCV), along with other members of the polyomavirus family, encodes a class of highly conserved proteins, T antigens, that are capable of inducing aneuploidy in cultured cells. We have previously isolated T-antigen DNA variants of JCV from both colon cancer tissues and the corresponding nonneoplastic gastrointestinal tissues, raising new questions about the role of JCV in the development of chromosomal instability of the colon. Based on the sequence of the transcriptional control region (TCR), JCV can be classified as archetype or tandem repeat variants. Among the latter, Mad-1, the prototype virus first isolated from a patient with progressive multifocal leukoencephalopathy, is characterized by lacking the 23-and 66-bp sequences that are present in the archetype and by duplication of a 98-bp sequence. In this study, we evaluated differences in the TCR of JCV isolated from colon cancer tissues and nonneoplastic epithelium. To characterize JCV variants, we first treated eight pairs of DNA samples from colon cancers and noncancerous tissue with topoisomerase I and then amplified and cloned the JCV TCR. We obtained 285 recombinant clones from the JCV TCR, 157 from nonneoplastic samples, and 128 from colon cancer tissues. Of these clones, 262 spanned the length of the JCV Mad-1 TCR: 99.3% from nonneoplastic samples and 82.8% from colon cancer tissues. In sequencing 54 clones in both directions, we did not find archetype JCV either in the nonneoplastic tissue or in the cancer samples. From all colon cancer tissues, 18 clones had a deletion of one 98-bp tandem repeat. This deleted strain was not detected in any of the nonneoplastic tissues (14 versus 0% [ 2 ؍ 23.6; P < 0.001]). Our study demonstrates that the only JCV strain present in the human colon is Mad-1, and the variant with a single 98-bp sequence is found exclusively in the cancer tissues. This strain may be involved in the development of chromosomal instability.The human polyomavirus JC virus (JCV) infects a large proportion of the population worldwide (23). The initial infection is unapparent, but subsequently JCV establishes a latent infection, becoming reactivated when the immune system is impaired (8). The JCV genome is double-stranded, negatively supercoiled circular DNA that consists of early and late coding regions separated by two noncoding regions at both the 5Ј and 3Ј ends. Within the noncoding sequences is the transcriptional control region (TCR), which contains the promoter and enhancer for the early and late proteins; the intergenic region separates the 3Ј regions of T antigen and the capsid protein, VP1.T antigen is an oncogenic protein capable of transforming mammalian cells by interacting with cellular proteins such as p53 and the Rb family proteins (5, 21). T antigen also has ATPase and helicase activities, the latter of which may eventually contribute to chromosomal breakage and recombination (7). We have recently reported the presence of JCV T-antigen DNA sequences in nonneoplastic gastrointestinal tissues, colon cancer samples,...