The pet20-delta deletion in Saccharomyces cerevisiae causes diminished growth on media containing non-fermentable carbon sources when incubated at both above and below the 30 degrees C optimal growth temperature. Furthermore, the pet20-delta strain has a greatly reduced level of cytochrome c, especially at 37 degrees C. The pet20-delta strain was sensitive to high NaCl and CaCl2 concentrations, hydrogen peroxide, oligomycin, polymixin B, amphotericin B and fluconazole. Biochemical fractionation and immunofluorescence staining demonstrated that Pet20p is located primarily in the mitochondria. Rhodamine B staining of pet20-delta cells showed an altered mitochondrial staining, indicating the possible lack of the mitochondrial membrane potential. We suggest that PET20 encodes a protein required for proper assembly or maintenance of mitochondrial components, but does not serve an enzymatic role. It is also possible that Pet20p may constitute a non-catalytic subunit of an uncharacterized mitochondrial complex or serve as a transporter or a coupling factor.