For filoviruses, such as Ebolavirus and the closely related Marburgvirus, transcriptional regulation is poorly understood. The open reading frames (ORFs) that encode the viral proteins are separated by regulatory regions composed of the 3' nontranslated region (NTR) of the upstream gene, highly conserved transcription stop and start signals, and the 5'NTR of the downstream gene. The conserved transcription stop and start signals either overlap, or they are separated by intergenic regions (IGRs) of different lengths. To assess the contribution of the regulatory regions to transcription, we established bicistronic minireplicons in which these regions were flanked by upstream and downstream ORFs, the Ebolavirus leader and trailer regions, and by T7 RNA polymerase promoter and ribozyme sequences. We found that the individual viral regulatory regions differ in their ability to direct protein synthesis from the upstream or downstream ORFs. Deletion or modification of the NTRs, IGRs, or transcription stop and start signals affected protein expression levels to various extents; for example, 5'NTRs appear to affect efficient protein expression from the downstream ORF, whereas 3'NTRs seem to attenuate protein expression from the upstream ORF. Overall, our data suggest that the regulation of Ebolavirus protein levels is complex.