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The present study aimed to analyze the mycoflora and the occurrence of alternariol (AOH) and alternariol monomethyl ether (AME) in grain samples of sunflower during different stages of plant development in Nova Odessa, State of São Paulo, Brazil. The data obtained were correlated with the presence of fungi in soil, wind-dispersed fungi, and the predominant climatic conditions of the region where the experiment was carried out. Analysis of the mycoflora revealed the presence of Fusarium verticillioides and Alternaria alternata in 70% and 46% of the samples, respectively. The profile of wind-dispersed fungi also showed F. verticillioides as the most frequently isolated fungus (68%), although A. alternata was detected in 28% of samples. In soil, Penicillium was the most frequent species (49.9%), followed by F. verticillioides (47.7%) and A. alternata (10.9%). Regarding water activity, sunflower grains presenting a high frequency of isolation of F. verticillioides and A. alternatahad a water activity ranging from 0.92 to 0.96, and statistical analysis revealed a negative linear correlation between the isolation of fungi and water activity. HPLC analysis showed that 18% of the sunflower grains were contaminated with alternariol (24.9-170.9 ng/g) and 10% with alternariol monomethyl ether (14.1-108.6 ng/g). The contamination of sunflower grains with AOH and AME in the field was low when compared to the LD50 necessary to cause toxicity to animals. However, the contamination with other toxigenic fungi such as F. verticillioides may indicate the presence of other mycotoxins in sunflower grains and a possible synergistic effect between them. This is the first report of the natural occurrence of alternariol and alternariol monomethyl ether in sunflower grains in Brazil.