To study the effect of time elapsed from the moment of taking samples on lactoperoxidase system components, we analyzed the activity of the lactoperoxidase enzyme and the concentrations of thiocyanate and hydrogen peroxide in 46 individual samples of Manchega ewe milk. Samples were maintained at a temperature of 4 degrees C until analysis, which took place at 6, 12, 24, and 48 h after extraction. Decreases were observed in lactoperoxidase activity when the analyses were performed at 48 h and in the thiocyanate and hydrogen peroxide concentrations at 12 h compared with those carried out earlier. Consequently, when the components of the lactoperoxidase system or its antibacterial activity are studied, the time elapsed since the sampling commenced must be taken into account. Similarly, the time elapsed is important when carrying out bacterial counts or residue screening by microbiological methods, during which the lactoperoxidase system may interfere. To study the component changes in the lactoperoxidase system during lactation, samples obtained 15, 30, 45, 60, 75, 90, 105, 120, and 135 d postpartum from 48 Manchega ewes were used. Average lactoperoxidase activity, thiocyanate, and hydrogen peroxide concentrations were 3.46 U/ml, 6.89 mg/L, and 0.39 mg/L, respectively, with significant variations throughout lactation. The thiocyanate and hydrogen peroxide levels at different lactation stages seemed to be insufficient to activate the lactoperoxidase system. Nevertheless, this could be achieved by adding 5 mg/L of thiocyanate and 8 mg/L of hydrogen peroxide at any time during lactation.