In addition to the genes involved in the structure of the viral particle, the bovine leukemia virus (BLV) genome contains a region called X which contains at least four genes. Among them, the tax and rex genes, respectively, are involved in transcriptional and posttranscriptional regulation of viral transcription. Two other genes, R3 and G4, were identified after cloning of the corresponding mRNAs from BLV-infected lymphocytes. Although the function of the two latter genes is still unknown, they appear to have important roles, since deletion of them restricts viral propagation in vivo. In order to assess the oncogenic potential of the R3 and G4 proteins, we first analyzed their ability to immortalize and/or transform primary rat embryo fibroblasts (Refs). In this assay, the G4 but not the R3 protein cooperated with the Ha-ras oncogene to induce tumors in nude mice. It thus appears that G4 exhibited oncogenic potential in vitro. To extend these observations in vivo, the pathology induced by recombinant viruses with mutations in G4 and in R3 and G4 was next evaluated with the sheep animal model. Viral propagation, as measured by semiquantitative PCR, appeared to be reduced when the R3 and G4 genes were deleted. These observations confirm and extend our previous data underlining the biological function of these genes. In addition, we present the results of a clinical survey that involves 39 sheep infected with six different BLV recombinants. Over a period of 40 months, 83% of the sheep infected with a wild-type virus developed leukemias and/or lymphosarcomas. In contrast, none out of 13 sheep infected with viruses with mutations in G4 or in R3 and G4 developed disease. We conclude that in addition to its oncogenic potential in vitro, G4 is required for pathogenesis in vivo. These observations should help us gain insight into the process of leukemogenesis induced by the related human T-cell leukemia virus type 1.