Improper processing and storage of raw milk contaminated with Staphylococcus aureus at elevated temperatures can result in the production of staphylococcal enterotoxins, especially type A (SEA), which is most frequently associated with food poisoning outbreaks such as the large one in Osaka, Japan, in 2000. In this study, the characteristics of S. aureus growth and SEA production at various high temperatures in raw milk samples were studied using two raw milk samples naturally containing low and high levels of natural microflora. The optimal temperatures found for SEA production in the two milk types were as high as 40 and 44°C (range, 36 to 48°C), and SEA production was dependent on the initial dose of S. aureus. These high temperatures were close to that of the outbreak in Japan. Thus, it was concluded that temperature was critical for SEA production in raw milk. It was also observed that natural microflora in the milk samples considerably suppressed SEA production but not staphylococcal growth. On the other hand, the amount of toxin in most milk samples decreased after peaking during storage.