Fanconi anemia is a rare autosomal recessive disease characterized by developmental defects of the thumb and radius, childhood onset of pancytopenic anemia and increased risk of leukemia. At least five complementation groups (A-E) have been defined but only the FAC gene has been cloned. Cells can be assigned to complementation group C by direct mutation analysis. To facilitate the search for additional FA genes and to measure the frequency of complementation groups, we have established new genetically marked immortalized FA-A and FA-D fibroblast cell lines and show their usefulness as universal fusion donors. These reference FA cell lines facilitated somatic cell fusion analysis and enabled us to assign the complementation group in 16 unrelated FA patients from North America. The majority of patients, belong to FA complementation group A (69%), followed by FA-C (18%), FA-D (4%) and FA-B or FA-E (9%).