We have isolated 15 spontaneous mutants resistant to one or several antibiotics like chloramphenicol, erythromycin and spiramycin. We have shown by several criteria that all of them result from mutations localized in the mitochondrial DNA. The mutations have been mapped by allelism tests and by two- and three-factor crosses involving various configurations of resistant and sensitive alleles associated in cis or in trans with the mitochondrial locus omega which governs the polarity of genetic recombination. A general mapping procedure based on results of heterosexual (omega(+)x omega(-)) crosses and applicable to mutations localized in the polar segment is described and shown to be more resolving than that based on results of homosexual crosses. Mutations fall into three loci which are all linked and map in the following order: omega-R(I)-R(II)-R(III). The first locus is very tightly linked with omega while the second is less linked to the first. Mutations of similar resistance phenotype can belong to different loci and different phenotypes to the same locus. Mutations confer antibiotic resistance on isolated mitochondrial ribosomes and delineate a ribosomal segment of the mitochondrial DNA. Homo- and hetero-sexual crosses between mutants of the ribosomal segment and those belonging to the genetically unlinked ATPase locus, O(I), have been performed in various allele configurations. The polarity of recombination between R(I), R(II), R(III) and O(I) decreases as a function of the distance of the R locus from the omega locus rather than as a function of the distance of the R locus from the O(I) locus.