Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the delta antigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to be required for genome replication. Another genome type (termed the L genome) extends the reading frame by 19 amino acids as a result of a single base change. Replication of the S and L genomes was studied in cultured fibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in a codelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was also observed following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutational analysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame's extra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.