Hepatitis B viruses replicate via reverse transcription of an RNA intermediate. This RNA pregenome serves as mRNA and is packaged into capsids and reverse transcribed. Both processes require the interaction of the viral reverse transcriptase, P protein, with the 5'-proximal epsilon-signals on the pregenome. For epsilon of human hepatitis B virus (HBV), the presence of a functionally important stem-loop structure with a central bulge, part of which acts as template for a short primer of first-strand DNA synthesis, has been experimentally confirmed. Based on phylogeny and its functional similarities to epsilon, the D epsilon-signal of duck hepatitis B virus (DHBV) had been proposed to have a similar structure which does not, however, correspond to the most stable computer prediction. We have therefore experimentally determined the secondary structures of D epsilon and of the H epsilon-signal of heron hepatitis B virus which differs considerably from D epsilon in primary sequence yet interacts productively with DHBV P protein. Our data support an HBV epsilon-like structure for both D epsilon and H epsilon; in particular the bulge is highly conserved, in accord with its special function in replication. However, the apical loop in H epsilon is much enlarged suggesting that, by an induced-fit mechanism, both RNAs may adopt a new, probably similar conformation in the complex with P protein.