The direct role of nicotine in tobacco carcinogenesis is still controversial. Recently, DNA damage by nicotine has been demonstrated in isolated human tonsillar tissue cells. Presently, these effects were investigated using mini-organ cultures (MOC) of human nasal epithelia. Intact MOC were repeatedly exposed to 2 and 4 mM nicotine for 1 h on culture days 7, 9, and 11. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. DNA damage was examined by Comet assay either directly after exposure or following a 24-h recovery period. Cell viability was not reduced by any treatment. On day 7, 1 h exposure to 2 and 4 mM nicotine caused a significant dose-dependent 3.3- and 5.6-fold increase in DNA damage compared to solvent controls. Although there was no evidence of significant repair within 24 h recovery, DNA damage was not further increased by nicotine on days 9 and 11. After double and triple exposure to 4 mM nicotine a significant reduction in DNA damage following 24 h recovery was observed. In contrast, treatment with MNNG resulted in a highly significant and cumulative increase in DNA migration up to 110-fold compared to controls. During recovery periods, MNNG-induced DNA damage was significantly repaired, leading to a 1.5- to 1.8-fold reduction in DNA migration within 24 h. These results confirm genotoxic effects of nicotine on human nasal epithelia. Further studies are needed to explain the lack of cumulative DNA-damaging effects of nicotine and the absence of significant DNA repair. These studies should include a battery of assays with multiple end points.