Viral mRNAs from lesions containing human papillomavirus type 6 (HPV-6) have previously been mapped on the viral DNA but relatively little is known about the control of mRNA production, or whether the mapped RNA termini correspond to promoters. By analysis of run-off transcripts synthesized in vitro, primer extension and measurements of promoter activity in fragments of the viral DNA introduced into cells, we have identified three promoters in the early region of the HPV-6b genome. These are: (i) at the end of the long control region upstream of the E6 open reading frame; (ii) upstream of E7 and (iii) upstream of E1. The promoter upstream of E1 was the most active. These results contrast with results of similar assays with HPV-18, in which the strongest promoter was that controlling expression of the transforming genes E6 and E7. In addition, a novel promoter was detected close to E5a, upstream of the late genes.