A mutant has been isolated from the mouse cell line LM(TK-) which is stably resistant to the macrolide antibiotic, carbomycin. Mitochondrial protein synthesis in this mutant was carbomycin resistant and chloramphenicol sensitive. Fusions between carbomycin-resistant and -sensitive cells produced hybrids, most of which were sensitive to 10 microgram/ml carbomycin. At 7.5 microgram carbomycin/ml, the average population resistance is low initially but increases with time. Carbomycin-resistant cells were enucleated and fused with carbomycin-sensitive cells under a variety of selective regimes designed to allow growth of carbomycin-resistant cytoplasmic hybrids (cybrids). No transfer of carbomycin resistance via the cytoplasm was detected. Karyoplasts from carbomycin-resistant cells showed a low transfer of resistance to 7.5 microgram carbomycin/ml in karyoplast-cell fusions. Carbomycin resistance in this mutant is therefore most likely encoded in a nuclear gene.