The major regulation protein of human papillomavirus (HPV) transcription is the viral E2 protein. Previous studies have identified a variety of alternatively spliced mRNAs containing multiple open reading frames (ORFs) encoding the E2 protein of HPV type 16. In these mRNAs the E2 ORF is contained as an internal ORF. In the present study, the translational capacities of three mRNA species starting at the p97 promoter and containing the 880/2581, 880/2708 and 226/2708 splice junctions upstream of the E2 ORF were investigated. Partial cDNAs spanning the E2 ORF and the related upstream ORFs were synthesized and assessed for E2 protein translation in vivo, in COS cells, and in vitro, in cell-free systems. Results of these analyses indicated that E2 protein was translated from all three mRNAs. Translation efficiency of E2 from the natural polycistronic templates was lower compared with that from a synthetic monocistronic control. Translation from the d-type bicistronic template (226/2708) was more efficient than that from the a-type (880/2708) and a'-type (880/2561) polycistronic templates. Further investigation of the translation of proteins encoded by the ORFs preceding the E2 ORF showed that a- and a'-type templates served for translation mainly of E7 but also of E6I, while the d-type template served for translation of E6IV. Overall, the translation data support the suggestion that the corresponding mRNAs may function as polycistronic transcripts.