HPV-6vc subgenomic fragments were inserted into an enhancer-dependent expression vector for chloramphenicol acetyltransferase (CAT) and assayed for the presence of transcriptional enhancing elements. A transcriptional enhancing element was detected in the noncoding region (NCR) of the HPV-6vc viral genome when the CAT assays were performed in viral transformed human kidney cell lines (293 and 324K), in human cervical carcinoma cell lines (HeLa and Siha), and in bovine papillomavirus type 1 (BPV-1) transformed mouse cells (C127-53). The NCR region of the HPV-6b genome was only capable of enhancing transcription of the CAT gene in the HeLa cell line at a level one-third that of the HPV-6vc NCR. The HPV-6vc NCR enhancing activity in C127-53 cells was further stimulated by the addition of sodium butyrate to the growth medium. Localization of the DNA sequences in the HPV-6vc NCR responsible for enhancing transcription revealed two distinct enhancer elements. One element (HPV-6vc position 7218-7544) was active in the 293, HeLa, Siha, and C127-53 cells. The second enhancer element (HPV-6vc position 7544-7971) was only capable of stimulating transcription in HeLa, C127-53, and Siha cells. When the HPV NCR-CAT expression vectors were cotransfected with a competitor plasmid (pNCR75) into C127-53 or HeLa cells then transcriptional enhancement decreased, indicating competition of cellular factors which affect both segments of the HPV-6vc NCR.