Due to a deficiency in mitochondrial protein synthesis, Chinese hamster lung (CHL) cell mutant Gal- 32 does not grow in galactose or fructose. This report examines the nuclear or cytoplasmic inheritance of this single, recessive mutation. In a control experiment, fusion of Gal+TGSTK- cells with Gal- 32TGRTK+ cells resulted in tetraploid hybrids (as verified by karyotyping) that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (2/3) of the control hybrids grew in galactose as expected since Gal+ is dominant over Gal-. Fusion of Rhodamine 6-G treated Gal+TGSTK- cells with Gal- 32TGRTK+ cells resulted in Rhodamine 6-G-tetraploid hybrids that were selected in hypoxanthine/aminopterin/thymidine medium. The majority (7/12) of the Rhodamine 6-G-hybrids grew in galactose as expected for a nuclearly encoded gene considering that Rhodamine 6-G interferes with transmission of mtDNA but not nuclear DNA. Therefore, these results are compelling in their demonstration of the nuclear origin of the Gal- 32 mutation.