The study of the composition of human milk has attracted a lot of interest, since it represents the most suitable pattern of nutrients for the younger infant. In the present study, the concentration of iron, copper and zinc was measured in the colostrum of 50 Brazilian mothers. The samples were collected from the day of birth to the fourth day postpartum. The levels of trace elements were determined by total reflection X-ray fluorescence analysis (TXRF) with synchrotron radiation. The accuracy of the measurements was calculated by determining the concentration of the elements in a standard solution (ICP-multielement standard solution) resulting in values ranging from 3.7 to 6.1% for the elements to be considered. We obtained the following values (mean7S.D.): 1.7271.01 mg/L Fe; 0.547 0.29 mg/L Cu; 6.9772.82 mg/L Zn. The concentrations of the determined elements were comparable with previously reported colostrum values. We concluded that TXRF is an adequate technique for evaluating trace elements in colostrum, providing multielement analysis in a single measurement, and requiring no pre-concentration of the sample. The technique can be optimized in further studies in order to evaluate a wide range of trace elements in the human milk, in a greater number of colostrum samples.