In plastids, editing of an ACG codon to an AUG codon creates the translation initiation codon for the psbL and ndhD transcripts in tobacco. To identify the RNA segment required for psbL editing, chimeric kanamycin resistance genes were constructed containing psbL deletion derivatives, and tested in vivo for editing in transgenic plants. We report here that a 22 nucleotide segment is sufficient to direct efficient psbL editing, including 16 nucleotides upstream and five nucleotides downstream of the editing site. Mutation of the A nucleotide to a C upstream of the editing site completely abolished editing, while mutation of the downstream G to a C only reduced the editing efficiency. Out of the 22 nucleotide editing target sequence, the 16 upstream nucleotides were found to compete with the endogenous psbL transcript for a depletable trans-factor. To test whether editing of initiation codons involves a common trans-factor, a chimeric gene containing the ndhD editing site was expressed in tobacco plastids. As for psbL, editing of the ndhD site requires a depletable trans-factor. However, the ndhD trans-factor is distinct from that required for psbL editing. Distinct cissequences and trans-factor requirements for the psbL and ndhD editing sites indicate an individual recognition mechanism for the editing of plastid initiation codons.