Using molecular cloning techniques, a clone containing a 7-kb insert of Spiroplasma species BC3 DNA that hybridized strongly to total labeled Spiroplasma tRNA was identified. Sequence analysis of a portion of this recombinant plasmid identified a cluster of tRNA genes. The gene order was as follows: tRNACys-tRNAArg-tRNAPro-tRNAAla-tRNAMet-tRNAIle and a portion of tRNASer All the genes encode the 3'-terminal CCA and have very high A + T and relatively long intergenic regions. An RNA polymerase promoter site was found upstream of the tRNACys gene. The tRNA gene cluster found in Spiroplasma can be compared with a similar Bacillus subtilis gene cluster, which raises interesting questions concerning gene organization and transcription.