Most feed is poor in iodine and iodine supplementation of cow's diets must guarantee milk iodine concentrations for humans that contribute to prevention of the deficiency and minimize the risk of exceeding an upper limit of iodine intake. Five Holstein cows were fed four iodine doses (via Ca(Iota O(3))(2).6H(2)O). In four sequential 14-d periods, doses of 0.2 (basal diet), 1.3, 5.1, and 10.1 mg iodine kg(-1) diet dry matter (DM) were administered. Samples of milk were collected during each period; blood was also sampled from each cow for each iodine dosage. In an 18-d depletion period, a non-supplemented diet was provided. Iodine was determined by inductively coupled plasma-mass spectrometry. The iodine content of milk and serum reflected the iodine dosages in feed significantly. The levels for the four doses tested in milk were 101+/-32, 343+/-109, 1215+/-222, and 2762+/-852 microg iodine kg(-1). The total amount of iodine in milk per day was 30-40% of ingested supplemental iodine. Omitting additional iodine resulted in a short-term reduction of serum and milk iodine following an exponential decay function. The iodine supplementation of 0.5-1.5 mg kg(-1) diet DM represents the requirement of the cow, resulting in 100-300 microg iodine L(-1) milk, which optimally contributes to human supply. The maximum dietary levels of former and present EU legislations (10 and 5 mg iodine kg(-1) cow feed) increase the risk of iodine excess in humans.