The upstream regulatory regions of human papillomavirus (HPV) types 1, 6b, 7, 11, 16, and 18, bovine papillomavirus type 1, and cottontail rabbit papillomavirus were cloned into transcriptional enhancer assay plasmids which carry the simian virus 40 early promoter lacking its own enhancer and the bacterial gene encoding chloramphenicol acetyltransferase (EC 184.108.40.206) (CAT). Enhancer activity, reflected by CAT gene expression, was detected in all of the upstream regulatory regions tested only when the recombinants were cotransfected with plasmids which express an intact E2 open reading frame of HPV types 1 and 11 or bovine papillomavirus type 1. Each E2 protein stimulated the enhancer from the same virus and, to somewhat lesser degrees, also those from the heterologous viruses. Hence, the enhancer and the E2 protein are functionally conserved among papillomaviruses. There was some nonreciprocity in the extent of trans-activation in heterologous E2-enhancer interactions. Primer extension analyses demonstrated that the E2 proteins increased the abundance of CAT gene mRNA. Tandem multiplication of the HPV type 11 enhancer sequence dramatically increased its response to E2 stimulation; this is possibly relevant to the pathogenicity of papillomaviruses.