Akv1-99, a variant of Akv murine leukemia virus, induces B-cell lymphomas with nearly 100% incidence and a mean latency period of 12 months after injection into newborn NMRI mice. PCR amplification and sequence analyses of DNA flanking integrated proviruses revealed proviral insertion into the N-ras/unr (upstream of N-ras) locus in 2 out of 13 B-cell lymphomas, both of which appeared clonal by Southern blotting analysis. These two tumors showed increased expression levels of N-ras by Northern blotting, as did a third tumor shown by reverse transcriptase PCR to have a nonclonal provirus integration located in the same area. However, no significant changes in expression were observed when using a specific probe for the unr gene. All proviruses were integrated in the same transcriptional orientation as unr and N-ras genes. By promoter insertion, the two Akv1-99 proviruses integrated between exon -1 and exon 1 of N-ras gave rise to two different spliced products, whereas the provirus integrated into unr used only an exon skipping pattern. The absence of mutations of the N-ras codons 12, 13, 18, and 61 suggests that activation of the proto-oncogene is exclusively due to overexpression by retroviral promoter insertion, and furthermore, Northern blot analyses indicate that the expression of unr is unaffected by N-ras overexpression even in the case where the unr gene itself is the target of proviral insertion. Thus, altogether our findings indicate that overexpression of N-ras plays a role in development of murine leukemia virus-induced B-cell lymphomas, leaving the expression of the tightly linked unr gene unaltered.