AbstractTheDrosophila tko25tpoint mutation in the gene encoding mitoribosomal protein S12 produces a complex phenotype of multiple respiratory chain deficiency, developmental delay, bang-sensitivity, impaired hearing, sugar and antibiotic sensitivity, and impaired male courtship. Its phenotypic severity was previously shown to be alleviated by inbreeding and to vary with mitochondrial genetic background. Here, we show similarly profound effects conferred by nuclear genetic background. We backcrossedtko25tinto each of 2 standard nuclear backgrounds, Oregon R andw1118, the latter used as recipient line in many transgenic applications requiring selection for thewhiteminigene marker. In thew1118background,tko25tflies showed a moderate developmental delay and modest bang-sensitivity. In the Oregon R background, males showed longer developmental delay and more severe bang-sensitivity, and we were initially unable to produce homozygoustko25tfemales in sufficient numbers to conduct a meaningful analysis. When maintained as a balanced stock over 2 years,tko25tflies in the Oregon R background showed clear phenotypic improvement though were still more severely affected than in thew1118background. Phenotypic severity did not correlate with the expression level of thetkogene. Analysis oftko25thybrids between the 2 backgrounds indicated that phenotypic severity was conferred by autosomal, X-chromosomal, and parent-of-origin-dependent determinants. Although some of these effects may betko25tspecific, we recommend that, in order to minimize genetic drift and confounding background effects, the genetic background of nonlethal mutants should be controlled by regular backcrossing, even if stocks are usually maintained over a balancer chromosome.