We have validated the analysis of nucleoplasmic bridges (NPBs) and nuclear buds as biomarkers of genomic instability within the cytokinesis-block micronucleus assay in long-term lymphocyte cultures. Lymphocytes from 20 subjects were cultured in medium containing 12-120 nM folic acid for 9 days. Binucleate cells were scored for micronuclei (MN), NPBs and nuclear budding on day nine after 24h incubation in the presence of the cytokinesis inhibitor cytochalasin-B. Folic acid concentration was correlated significantly (P<0.0001) and negatively (r=-0.63 to -0.74) with all these markers of chromosome damage. Chromosome damage was minimised at 60-120 nM folic acid, which is greater than the concentration of folate normally observed in plasma (<30 nM). Current evidence suggests that (a) NPBs originate from dicentric chromosomes in which the centromeres have been pulled to the opposite poles of the cell at anaphase and are therefore, indicative of chromosome rearrangement and (b) that the nuclear budding process is the mechanism by which cells remove amplified DNA and is therefore a marker of gene amplification. The strong correlation between micronucleus formation, nuclear budding and NPBs (r=0.75-0.77, P<0.001) is supportive of the hypothesis that folic acid deficiency causes genomic instability and gene amplification by the initiation of breakage-fusion-bridge (BFB) cycles. These results also suggest that the CBMN assay may be a useful model for the study of the BFB cycle which may be one of the key mechanisms for the hypermutability phenotype required for the rapid evolution of cancer cells.