Fanconi anemia (FA) is a clinically and genetically heterogenous disease that is usually diagnosed on the basis of chromosomal instability reflecting the hypersensitivity towards the DNA cross-linking agents diepoxybutane (DEB) and/or mitomycin C. A less well-known cellular feature that characterizes FA patients is an intrinsic cell cycle disturbance consisting of prolonged progression through, and arrest within, the G2 phase compartment of the cell cycle. In a collaborative blind study, we have evaluated 72-hour lymphocyte cultures from 66 patients with clinical suspicion of FA both for DEB sensitivity and cell cycle disturbance. A concordant result was obtained in 63 of 66 cases. Each of the 3 discordant, but only 1 of the concordant cases presented with overt leukemia. Seventeen cases were identified as classical FA because of their increased DEB sensitivity and G2 phase blockage. Five cases showed a cell cycle disturbance but only borderline DEB sensitivity. These cases might represent atypical or nonclassical forms of FA. They would have been missed by cell cycle studies without concomitant DEB testing. Used in conjunction, cytogenetic and flow cytometric testing provide for the currently optimal diagnosis of FA in nonleukemic patients.