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Purpose: The effect of one pesticide spraying season on DNA damage was measured on B and T lymphocytes among open-field farmers and controls. Methods: At least two peripheral blood samples were collected from each individual: one in a period without any pesticide application, several weeks after the last use (January, at period P0), and another in the intensive pesticide spraying period (May or June, at period P4). DNA damage was studied by alkaline comet assay on isolated B or T lymphocytes. Results: Longitudinal comparison of DNA damage observed at both P0 and P4 periods revealed a statistically significant genotoxic effect of the pesticide spraying season in both B ( P = 0.02) and T lymphocytes ( P = 0.02) in exposed farmers. In contrast, non-farmers did not show any significant modifications. DNA damage levels in B and T lymphocytes were significantly higher in farmers than in non-farmers during the P4 period ( P = 0.003 and P = 0.001 for B and T lymphocytes, respectively) but not during the P0 period. The seasonal effect observed among farmers was not correlated with either total farm area, farm area devoted to crops or recent solar exposure. On average, farmers used pesticides for 21 days between P0 and P4. Between the two time points studied, there was a tendency for a potential effect of the number of days of fungicide treatments ( r = 0.43; P = 0.11) on T lymphocyte DNA damage. Conclusions: A genotoxic effect was found in lymphocytes of farmers exposed to pesticides, suggesting in particular the possible implication of fungicides. [ABSTRACT FROM AUTHOR]