A simple and reliable cytogenetic test for Fanconi's anemia (FA) that is based on the hypersensitivity of FA cells to mitomycin C (MC) is described. Equal volumes of whole blood from a patient in whom the diagnosis of FA is suspected and from a normal person of the opposite sex are co-cultured in phytohemagglutinin-containing medium in the presence and absence of MC. After five days' co-cultivation, 100 quinacrine-stained metaphases from both the MC-containing and the MC-free cultures are examined for the presence of a Y chromosome using fluorescence microscopy. In all bona fide FA patients in whom testing was successful, hypersensitivity to MC was readily demonstrated by the striking deficiency of FA metaphases (0.9% to 14.9%) in the MC-containing co-cultures. In contrast, none of the three patients with Diamond-Blackfan anemia and none of the five with undiagnosed conditions reminiscent of FA exhibited hypersensitivity to MC; cells from them, from parents of FA patients, and from several normal laboratory personnel constituted approximately half of the metaphases (40.4% to 71.2%) of MC-containing co-cultures, as would be expected in the absence of hypersensitivity to MC.