SYNOPSIS.The 16S ribosomal RNA of the chloroplast ofEuglena gracilisstrain Z has been characterized in terms of its 2‐dimensional electrophoretic “fingerprint” (T1 ribonuclease). Over 100 spots were resolved on the “fingerprint” and each spot was characterized as to which RNA oligonucleotide fragment(s) it contained. When compared to similar analyses of prokaryotic 16S rRNAs and eukaryotic cytoplasmic 18S rRNAs, the chloroplast 16S rRNA was a typically prokaryotic RNA, but bore little if any relationship to eukaryotic 18S rRNAs. Therefore, the cistrons for chloroplast 16S rRNA are related to the equivalent prokaryotic cistrons, but, apparently, are not related to the equivalent eukaryotic cistrons. Among the organisms available for comparison, theEuglenachloroplast 16S rRNA appears most closely related to the 16S rRNA of the eukaryote,Porphyridium cruentum(a red alga), and at least distantly related to the 16S rRNAs of the blue‐green algae and perhaps also to