Bovine papilloma virus-transformed hamster embryo fibroblasts (HEF-BPV) reacted to exposure to tricyclodecan-9-yl-xanthogenate (D609) with immediate reversion to the growth kinetics and the flat morphology of the untransformed parental cells. After six population doublings in the presence of D609, clones which displayed an untransformed morphology in the absence of D609 arose with a high frequency (90%). Such clones had reacquired a limited in vitro lifetime and had lost the ability to induce tumors in athymic nude mice. At the molecular level the revertant clones had lost all extrachromosomal monomeric BPV-1 DNA molecules. Only high molecular weight (HMW) oligomeric BPV-1 DNA that was probably integrated into the cellular genome was still detectable in a methylated transcriptionally inactive state. In contrast to transformed cells, the revertant clones no longer transcribed BPV-1-specific mRNA molecules, but were stimulated by a tumor promoter to transient viral gene expression. This article provides direct evidence for the complete reversibility of the property of "immortality".