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There is a need for a rapid assay to identify agents that damage mitochondria because the mitochondrion may be an important target for numerous environmental mitotoxins. Certainly at least one chemotherapeutic regimen (CHOP therapy) that includes doxorubicin can induce cardiomyopathy through mitochondrial genotoxicity in cardiac muscle cells. Yeast cells (1.5 × 10⁶-10⁷) in water are spread on a YEPD plate, and, when the suspension of cells has dried, a small well (12 mm diameter) is cut into the agar; 200–400 μl of a solution of the presumptive mitochondrial genotoxin is placed in the well, and the plates are incubated for 2 days. The genotoxin forms a concentration gradient through the agar and affects the growing cells. An overlay containing tetrazolium chloride is added, and the plates are incubated for 6–24 hr. Respiring cells turn red, and nonrespiring cells, with damaged DNA or inhibited respiratory chains, that are adjacent to the well, are white. A white ring, or a more lightly colored red ring, around the well indicates the presence of cells with lowered respiratory activity which may be fully reversible when the mitochondrial genotoxin is removed. In preliminary experiments, doxorubicin (= adriamycin) shows strong activity with this assay; cyclophosphamide is negative, and 4-hydroxycyclophosphamide, a metabolite of cyclophosphamide, is weakly positive. Ethidium bromide, methotrexate, 5-fluorouracil, and 5-fluorocytosine also are mitochondrial genotoxins. Antifungal agents similar to 5-fluorocytosine and anthelmintic compounds such as pyrvinium iodide can be powerful mitochondrial genotoxins. Environ. Mol. Mutagen. 38:153–158, 2001 © 2001 Wiley-Liss, Inc.