Mitochondrial transfer RNA genes have been ordered relative to the position of five mitochondrial drug resistance markers, namely, chloramphenicol (C),1 erythromycin (E), oligomycin I and II (OI, OII), and paromomycin (P). Forty-six petite yeast clones that were genetically characterized with respect to these markers were used for a study of these relationships. Different regions of the mitochondrial genome are deleted in these individual mutants, resulting in variable loss of genetic markers. Mitochondrial DNA was isolated from each mutant strain and hybridized with eleven individual mitochondrial transfer RNAs. The following results were obtained: i) Of the seven petite clones that retained C, E, and P resistance markers (but not O1 or O11), four carried all eleven transfer RNA genes examined; the other three clones lost several transfer RNA genes, probably by secondary internal deletion; ii) Prolyl and valyl transfer RNA genes were located close to the P marker, whereas the histidyl transfer RNA gene was close to the C marker; iii) Except for a glutamyl transfer RNA gene that was loosely associated with the O1 region, no other transfer RNA genes were found in petite clones retaining only the O1 and/or the OII markers; and iv) Two distinct mitochondrial genes were found for glutamyl transfer RNA, they were not homologous in DNA sequence and were located at two separate loci. The data indicate that the petite mitochondrial genome is the result of a primary deletion followed by successive additional deletions. Thus an unequivocal gene arrangement cannot be readily established by deletion mapping with petite mutants alone. Nevertheless, we have derived a tentative circular map of the yeast mitochondrial genome from the data; the map indicates that all but one of the transfer RNA genes are found between the C and P markers without forming a tight cluster. The following arrangement is suggested: -P-pro-val-ile-(phe, ala, tyr, asp)-glu2- (lys-leu)-his-C-E-O1-glu1-OII-P-.