The upstream regulatory region (URR) of bovine papillomavirus type 1 (BPV) contains promoters and a conditional transcriptional enhancer that is trans-activated by the viral E2 protein. After deleting the 5` and 3` ends of BPV URR, BamHI linkers were inserted into several positions of BPV URR without causing an addition or a deletion of URR sequences. Most linker scanning mutations did not show any effects on the transcription of P7940 and P89 promoters in BPV URR. However, several mutants showed reduced transcriptional activities. Based on our results we found that the AP-2 and Spl binding sites were important for basal level transcription of BPV URR in the absence of the E2 protein and that the CTF/NF-1 site is dispensable for E2 tmnsactivation of BPV URR transcription.