The aim of this study was to assess levels of aflatoxin M1 (AFM1) in milk samples from Kerman, Iran. AFM1 was detected in 72 samples, ranging in concentration from <0.01 to 0.41 µg l(-1). The samples were analyzed using immunoaffinity column for clean-up and HPLC for determining AFM1. Milk samples were collected from six dairy farms. AFM1 was found in ∼ 50% of the milk samples. The average level of AFM1 was below the tolerance limit (0.05 µg l(-1)), but 50% of the samples had greater levels than the maximum tolerance limit accepted by EU and the Iranian national standard. The method detection limit and limit of quantification were 0.01 and 0.03 µg l(-1), respectively, and recovery of the method was 87%. The results showed that AFM1 contamination is a serious problem for public health. To achieve a low level of AFM1 in milk, cattle feed must be monitored regularly for aflatoxin contamination and protected from fungal contamination as much as possible.