Restriction fragments containing the 16S rRNA gene of the western aster yellow mycoplasmalike organism (SAY-MLO) were identified in Southern blots probed with cloned fragments of the western X-disease mycoplasmalike organism 16S rRNA gene. Two fragments which contained the entire SAY-MLO 16S rRNA gene and flanking DNA were cloned in M13 and sequenced. The SAY-MLO 16S rRNA gene is approximately 1,535 bp long, has a G+C content of 47 mol%, and has an overall secondary structure similar to that proposed for Escherichia coli. Putative rRNA promoter sequences and sequences involved in processing of the primary rRNA transcript were similar in the SAY-MLO, two Mycoplasma species, and Bacillus subtilis, suggesting that these prokaryotes and the mycoplasmalike organisms may have similar transcriptional and processing enzymes. We identified two tRNA genes, a tRNA(Tyr) (GTA) gene upstream from the 16S rRNA gene and a tRNA(Ile) (GAT) gene in the spacer region between the 16S and 23S rRNA genes. Comparisons of the SAY-MLO 16S rRNA nucleotide sequence with 16S rRNA sequences of other organisms indicated that the SAY-MLO is phylogenetically related most closely to other plant-pathogenic mycoplasmalike organisms, followed by Anaeroplasma species, Acholeplasma species, and some Mycoplasma species.