We have determined the entire genomic sequence of the Pennsylvania strain, which was isolated along with the Virginia strain during the emergence of Ebola virus Reston in 1989/90 in the United States. Thus, either the Pennsylvania or Virginia strain, neither of which had been previously molecularly characterized, can be considered as the prototype for Ebola virus Reston. Comparative analysis showed a high degree of homology to the concomitantly analyzed and recently published Philippine strain of EBOV Reston from 1996 (Ikegami et al., Arch. Virol., 146 (2001) 2021). In comparison to EBOV Zaire, strain Mayinga, conservation could be found within the open reading frames, the 3' leader and 5' trailer region and the transcriptional signals, whereas the non-coding and intergenic regions did not show any homology. This clearly supports that EBOV Reston is a distinct species within the genus Ebola-like virus but which seems to be similar to other members with respect to transcription and replication strategies. The sequence determination provides the basis for the development of a reverse genetics system for Ebola virus Reston, which is needed to study differences in pathogenicity among filoviruses.