Fanconi anemia (FA) is a chromosome instability syndrome. Chromosomal aberrations (CA) are mainly of the chromatid-type suggesting that a deficiency in post-replicative repair may be involved. Our objective was to study whether inhibition of deoxyribonucleotide (dNTP) synthesis during the G2 phase by hydroxyurea (HU) produces an increase in CA in FA cells. Lymphoblastoid cell lines from a normal subject and four FA patients of types A to D were grown following standard protocols. Half of the cultures were treated with 10 ng/ml of mitomycin C (MMC) for 24 h and half served as controls. Three hours before harvesting, the cultures were treated with 2mM HU or 0.25 mM caffeine to inhibit post-replicative repair. Using this protocol only cells that were in G2 during HU or caffeine treatment are in metaphase at the time of harvest. Fifty metaphases per cell line and treatment were analyzed. Each culture was made in triplicate and Chi square analysis was used to compare the data. In all cell lines, caffeine induced a significant increase of CA only when the cells were pretreated with MMC. This increase was additive with respect to the independent caffeine and MMC treatments. HU alone induced a significant increase of CA only in FA cell lines (p<0.05). Following a pretreatment with MMC, HU induced an additive increase of CA in all cell lines except in FA-A where a synergistic effect was observed (three times higher, p<0.05). These results show that HU interferes with G2 DNA repair activity in FA cell lines in a heterogeneous way. FA-A was the most sensitive, suggesting that this cell line requires a proficient postreplicative repair and an ample supply of dNTPs. We are now studying whether HU-induced CA are due to depletion of dNTPs or generation of free radicals.