Fanconi anemia (FA) is a heterogeneous autosomal recessive DNA repair disorder with at least 11 complementation groups (ABCD1D2EFGIJL) characterized by mutations in 8 genes (ACD1D2EFGL) identified so far. This large number of genes affected in combination with extreme difficulties to identify the mutations in FA-A and -D2, which when mutated account for more than 60% of patients, prompted us to develop a rapid laboratory procedure that is capable of subtyping the FA complementation group in primary T cells (Hanenberg et al. 2002). In order to determine the sensitivity and specificity of this approach, we prospectively analyzed whether PB-derived T cells from 164 FA patients with proven FA could be used to classify the patients as FA-A/C/F/G, or FA-nonACFG. To this end, depending on the amount of PB available and the growth kinetics of the T cells, PB-MNCs were prestimulated on CD3/CD28-coated plates and then transduced in the presence of IL2 on CH296-coated plates with oncoretroviral vectors expressing either the FANCA/C/F/G or the EGFP cDNA as marker gene off the retroviral LTR. Transduced cells were exposed to increasing amounts of mitomycin C (MMC) as DNA crosslinking agent and then analyzed 4–6 days later for survival by flow cytometry as indicator for successful correction of the cellular FA phenotype. Successful complementation was defined as clear reduction of the in vitro hypersensitivity to MMC with one specific oncoretroviral vector occuring at at least three different MMC concentrations. The complementation group assignment was confirmed by mutation analysis, FANCD2 western blotting or successful correction of primary fibroblasts or EBV-immortalized B-cell lines after transduction with NEO containing retroviruses (ACD2EFG) followed by G418 selection. Despite proven FA, 27 samples from 164 patients (16.5%) showed no growth arrest in response to MMC and therefore could not be subtyped (15 FA-A, 2 C, 8 nonACFG, 2 FA patients of unknown type). Therefore, the T cell somatic mosaicism was below 20% in our study. The correct complementation group was identified in 116 out of 137 patients (85%) with MMC hypersensitivity (78 A, 8 C, 2 F, 15 G, 13 nonACFG). Analyses on 21 samples (9 A, 3 C, 2 G, 3 nonACFG, 4 unknown) out of 137 samples (15%) failed due to ‘no growth', bacterial/fungal contamination of cultures or technical reasons including low gene transfer. The results from the retroviral complementation group subtyping by cellular correction was correct in all cases where both pathogeneic germline mutations were known demonstrating that no dominant negative mutations occurred in the four FA genes FANCA/C/F/G in the patient population studied. In summary, this prospective study establishes retroviral complementation group analysis as a quick and reliable assay for identifying the affected gene in the vast majority of FA patients with a sensitivity of 85% and a specificity of 100%. Overexpression of the wild-type gene via retroviral vectors in cells from patients with genetic disorders and subsequent analysis of the cellular phenotype is suitable for all diseases where the phenotype is accessible for test in the laboratory.