The ras proteins (Harvey, Kirsten and N-ras) are key regulators of signal transduction and a perturbation of their GDP/GTP cycle is frequently observed in tumors. In mammals, N-ras constitutes with unr (upstream of N-ras) a tightly linked tandem of ubiquitously expressed genes. Although unr and N-ras appear to be involved in distinct functions, this unusual genetic organization could be important for the regulation of N-ras expression. Specifically, transcription of unr could negatively regulate that of N-ras by transcriptional interference. To investigate this possibility, we have deleted the unr promoter by homologous recombination in murine embryonic stem cells. Analysis of tissues of heterozygous mice revealed an increase in N-ras mRNA accumulation ranging between 20 and 65%, in agreement with the suppression of a transcriptional interference.