Two forms of Mod5p, a tRNA modification enzyme, are found in three intracellular compartments, mitochondria, cytoplasm and nucleus, but are encoded by a single MOD5 gene. The two forms of the enzyme, Mod5p-I and Mod5p-II differ at the N-termini and are produced by initiation of translation at different start codons. Mod5p-I does contain a mitochondrial targeting signal and is distributed between mitochondria and cytoplasm, whereas Mod5p-II is found in the cytosol and nucleus (Boguta, M., et al. 1994, Mol. Cell. Biol. 14, 2298-2306). In the present work mutants which mislocalize the Mod5p-I enzyme were isolated. The screen was based on a correlation between the amount of cytosolic protein and the efficiency of tRNA mediated suppression. Identification of mutants is possible because a red pigment accumulates in the cells unable to suppress an ade2-1 nonsense allele. The maf1 mutant, with an altered intracellular localization of the Mod5p-I protein, was isolated. Immunofluorescence data suggest that the mutation causes mislocalization of the Mod5p-I to the nucleus.