We have developed an in vitro transcriptional assay using Escherichia coli RNA polymerase to initiate the replication of peach latent mosaic viroid (PLMVd). Regardless of the polarity of the PLMVd strand used as template, initiation in vitro occurred at the same hairpin structure. These initiation sites correspond to the 5'-ends of two small (280 nt) PLMVd-related RNAs found in infected peach leaves. Using a series of truncated PLMVd-derived transcripts, we have demonstrated that the viroid domain composed solely of the self-complementary hammerhead sequences is sufficient to trigger polymerase-driven replication in vitro. These data suggest that the bacterial-like RNA polymerase from peach chloroplasts catalyzes PLMVd replication.