The DNA sequence requirements of two T4 tertiary replication origins have been characterized by a deletion analysis of origin-containing plasmids. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 base pairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.