Mitochondrial biogenesis and function depend upon the interaction of mitochondrial and nuclear genomes. Forward genetic analysis of mitochondrial function presents a challenge in organisms that are obligated to respire. In the S-cytoplasmic male sterility (CMS-S) system of maize, expression of mitochondrial open reading frames (orf355-orf77) conditions collapse of developing haploid pollen. Nuclear restorer-of-fertility mutations that circumvent pollen collapse are often homozygous lethal. These spontaneous mutations potentially result from disruption of nuclear genes required for mitochondrial gene expression, in contrast to homozygous-viable restorer-of-fertility alleles that function to block or compensate for the expression of mitochondrial CMS genes. Consistent with this hypothesis, the homozygous-lethal restoring allele historically designated RfIII was shown to be recessive in diploid pollen produced by tetraploid CMS-S plants. Accordingly, the symbol for this allele has been changed to restorer-of-fertility  lethal  1 (rfl1). In haploid rfl1 pollen, orf355-orf77 transcripts and mitochondrial transcripts encoding the α-subunit of the ATP synthase (ATPA) were decreased in abundance. Haploid rfl1 pollen failed to accumulate wild-type levels of ATPA protein, indicating that functional requirements for mitochondrial protein accumulation are relaxed in maize pollen. The CMS-S system and rfl mutations therefore allow for the selection of nuclear mutations disrupting mitochondrial biogenesis in a multicellular eukaryote.