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The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) was investigated on the induction of chromosomal aberrations (CA) in cultured bovine peripheral lymphocytes. Cultures from two healthy donors were treated with tolylfluanid-based fungicide at concentrations ranging from 1.7 to 17.5 μg/ml for the last 24 and 48 hours of cultivation. Conventional cytogenetic method (CA assay) with Giemsa staining as well as fluorescence in situ hybridization (FISH) with whole bovine chromosomes 1 and 5 painting probes were used in the experiment. In the CA assay, no clastogenic effect of the fungicide was found after Euparen Multi treatment for 24 hours. On the contrary, significant elevation in polyploidy induction was observed with dose-dependence in one of the donors. Using prolonged time of exposure to the fungicide (the last 48 h of the cultivation), a slight clastogenic effect was detected at the doses of 8.75 and 17.5 μg/ml (P < 0.05, P < 0.01, respectively) in donor 1 and at the dose of 8.75 μg/ml (P < 0.05) in donor 2. The highest doses tested caused reduction of the mitotic indices (MI) (P < 0.05, P < 0.01) in both donors as well as both treatment times. The evaluation of stable structural aberrations in lymphocytes by two-colour FISH (48 h exposure) using bovine chromosome painting probes revealed the presence of nonreciprocal translocations at two examined concentrations (3.5 μg/ml and 8.75 μg/ml).