Infection of CV-1 monkey cells with SV40-GBM, a papovavirus isolated from a human glioblastoma multiforme, resulted in the appearance of defective viral DNA molecules. In contrast to SV40 wild-type, two main types of variant DNA molecules could be found after three viral passages at multiplicities of infection of about 10. The molecules of one variant DNA (GBM3-L) were about 19% shorter than the GBM3-H DNA molecules and the DNA of the original GBM isolate, as demonstrated by electron microscopy. Restriction enzyme analysis revealed that GBM3-L DNA had lost both the EcoRI and the HpaII cleavage sites which are located in the late viral genome region. Furthermore, SV40 GBM3-L did not possess the two PvuII sites which are located in the late genome region, and a portion of the GBM3-H and GBM3-L DNA molecules had lost the unique KpnI site. Heteroduplex analysis verified that the rearrangements in the GBM3-L DNA are located only in the late region of this DNA. The possible differences between SV40 wild-type and SV40-GBM are discussed on the basis of these results.