A genetic analysis of the resistance phenotype of a recently described chloramphenicol-resistant variant derived from the human cell line, HeLa (MC63), has been undertaken. Whole cells or enucleated fragments, produced by treatment with cytochalasin B, were fused with chloramphenicol-sensitive mouse, or human cells. Enucleated cells (cytoplasts) act as very efficient donors of the resistance phenotype in fusions with other human cell lines derived from HeLa. We conclude that chloramphenicol resistance is determined cytoplasmically. Transfer of resistance to unrelated human cell lines occurred at much lower frequency and we were unable to demonstrate transfer to mouse cells. An examination of mitochondrial protein synthesis in the fusion products of cytoplasts and whole cells suggested that mixed populations of mitochondria from both parental cells were maintained under the conditions of selection.