The chloroplast genome of Euglena gracilis contains a supplementary gene for a 16 S rRNA (s16 S rrn gene), which is not part of a complete rrn operon. An open reading frame (ORF406) is located downstream of the s16 S rrn gene. Chloroplast RNA was hybridized with cloned DNA fragments of this region and the hybrids were analysed by electron microscopy and S1-nuclease protection experiments. The s16 S rrn gene and the ORF406 are transcribed as one continuous 3.6 kb long RNA, which starts just upstream of the 5'-end of the s16 S rrn gene. The 3'-end occurs at multiple sites within a region of 700 bases downstream of the ORF. Northern blot analysis shows that the abundance of the transcript is comparable with that of other chloroplast mRNAs.