Investigations of the structure and expression of the N-ras gene in mammals has led to the identification of another gene designated unr, which is located immediately upstream of N-ras. These two genes are transcribed in the same orientation and the intergenic distance is of the order of 150 nucleotides. This genetic organization has been observed in the genome of guinea pig, rat, mouse and man with a very high level of sequence conservation in the intergenic region. This unusual gene clustering suggests that the transcriptional regulation of this locus could involve common regulatory sequences as well as transcriptional interference between the two genes. In this study, we have isolated and characterized the human unr promoter. A cluster of transcription initiation sites was mapped by primer extension and RNase protection and shown to be located in a CpG island devoid of TATA and CAAT boxes. Functional organization of the promoter was investigated by measuring the ability of a set of 5' deletions within a1 kb promoter region to drive the expression of the luciferase gene. These studies indicated a very strong promoter activity in NIH 3T3 cells and the presence of positive and negative regulatory domains. Nevertheless, a 90 bp fragment showed the same level of promoter activity as the 1 kb fragment. We also showed that ras genes can transactivate the unr promoter activity and that the 90 bp fragment responded to this transactivation.