To investigate the organization of tRNA genes in the honeybee pathogen, Spiroplasma melliferum (previously referred to as Spiroplasma sp. BC3), total labeled tRNA from S. melliferum was used as a hybridization probe to an EcoRI digest of the genomic DNA. The results show two, or possibly three, strongly hybridizing bands. Comparing the pattern obtained to that of Bacillus subtilis DNA suggests either that the S. melliferum tRNA genes are more extensively clustered or, more likely, that the S. melliferum genome does not encode a full complement of tRNA genes, as has been suggested for other Mollicutes. We screened a library of EcoRI fragments of S. melliferum DNA cloned in pBR322 with total radioactive tRNA as a probe and selected one of the tRNA gene clusters. Subsequent sequence analysis of a portion of the clone showed 10 tRNA genes probably comprising a single operon. Comparison of sequence with a tRNA gene cluster from Mycoplasma mycoides and a portion of a cluster from B. subtilis showed an identical order of tRNA genes and the isoacceptors encoded. Such a striking comparison in gram-positive eubacteria suggests an important function for regulation and co-transcription of these tRNA genes.