In vitro transcription was used to show that RNA polymerase III is responsible for the initiation of transcription at a position 200 bp upstream from the epsilon-globin major cap site. High levels of -200 transcription interferes with the RNA polymerase II major cap site transcription. Using DNA mediated transient expression, the ratio of -200 to +1 transcription can be modulated by either the direction of replication or the presence of an enhancing element in the vector. We suggest that this heterogeneous usage of cap sites is not related to epsilon-globin gene transcription in vivo, but is instead the result of a combination of factors inherent to transient expression experiments.