Increasing use is being made of colorimetric assays to quantitate viable cells, e.g., the cellular reduction of the tetrazolium salt, MTT, to formazan by mitochondrial succinate dehydrogenase. We validated this assay for cell proliferation in mixed lymphocyte cultures as compared with 3H-thymidine uptake, and for inhibition of cell proliferation induced by interferon with results compared by direct cell counting. We also found that cells do not, as previously assumed, require functional mitochondria: there were no differences in formazan production by normal cells or respiratory defective cells in which mitochondria had been poisoned by the nucleic acid toxin, ethidium bromide. The high reproducibility of MTT metabolism by lymphoblasts and various cultured cell lines establishes the reliability and versatility of this method for quantitating cell numbers.