Aflatoxin M1 (AFM1) distribution in curd, whey, Manchego cheese, the traditional Spanish whey cheese Requesón and Requesón whey, and its stability during two different cold treatments, have been studied. Raw ewe’s milk was artificially contaminated with AFM1 in a final concentration of 50 and 100 ng kg-1, and was used to produce Manchego cheese. AFM1 determinations were carried out by HPLC with fluorimetric detection after immunoaffinity clean-up. The mean AFM1 concentrations in the produced curd and Manchego cheese were approximately 2- and 3-fold higher than the initial milk they were made from, and the levels of this toxin remaining in whey were 42.3 % and 51.3 % of the initial concentrations. In the Requesón samples, the mean AFM1 values were 1.7 times higher than those in the corresponding whey, while 33.7 % and 44.4 % of the AFM1 concentration detected in milk also appeared in the Requesón whey. Short refrigeration and freezing periods did not affect the toxin levels in either curd or Requesón samples. When ewe’s milk destined for Manchego cheese-making is AFM1-contaminated at the EU limit level (50 ng kg-1) or double, a concentration of this toxin will appear in the manufactured products, but values will be considerably below the toxic doses (Tolerable Daily Intake = 2 ng kg-1 body weight per day), which poses a human health problem.