The chloroplast mRNAs from Euglena gracilis fall into two classes. One group of mRNs from this organelle contains a Shine-Dalgarno sequence 5' to the start codon, while the other group of mRNAs does not have a conserved sequence signal in the 5'-untranslated region. To investigate the start signals for E. gracilis chloroplast mRNAs that do not carry a Shine-Dalgarno sequence, 30 S initiation complex formation has been studied using a series of transcripts carrying the wild-type translational start site of ribulose-bisphosphate carboxylase/oxygenase (rbcL) or mutated derivatives of this site. Mutation of the start codon of the rbcL gene indicates that the chloroplast 30 S subunit is recognizing only the correct AUG codon. The analysis of the messages from a series of deletion mutants shows that a minimum of delta 20 residues 5' to the AUG codon is required for activity. Maximal activity requires the full 55-base leader sequence. Surprisingly, a transcript carrying the inverse complement of 48 bases in the leader is delta 60% as active as the wild-type message in promoting initiation complex formation. Introduction of a Shine-Dalgarno sequence in the 5'-leader increases the activity of the mRNA only delta 1.4-2-fold. The presence of an oligodeoxynucleotide containing a strong Shine-Dalgarno sequence does not significantly inhibit the formation of initiation complexes at the rbcL start site. Similar results are obtained when initiation complexes are formed with initiation factors from either E. gracilis chloroplasts or Escherichia coli.