Iodine intake is important for thyroid function. Iodine content of natural waters is high in some areas and occurs bound in humic substances. Tap water is a major dietary source but bioavailability of organically bound iodine may be impaired. The objective was to assess if naturally occurring iodine bound in humic substances is bioavailable. Tap water was collected at Randers and Skagen waterworks and spot urine samples were collected from 430 long-term Randers and Skagen dwellers, who filled in a questionnaire. Tap water contained 2 μg/l elemental iodine in Randers and 140 μg/l iodine bound in humic substances in Skagen. Median (25; 75 percentile) urinary iodine excretion among Randers and Skagen dwellers not using iodine-containing supplements was 50 (37; 83) μg/24 h and 177 (137; 219) μg/24 h respectively (P < 0·001). The fraction of samples with iodine below 100 μg/24 h was 85·0 % in Randers and 6·5 % in Skagen (P < 0·001). Use of iodine-containing supplements increased urinary iodine by 60 μg/24 h (P < 0·001). This decreased the number of samples with iodine below 100 μg/24 h to 67·3 % and 5·0 % respectively, but increased the number of samples with iodine above 300 μg/24 h to 2·4 % and 16·1 %. Bioavailability of iodine in humic substances in Skagen tap water was about 85 %. Iodine in natural waters may be elemental or found in humic substances. The fraction available suggests an importance of drinking water supply for population iodine intake, although this may not be adequate to estimate population iodine intake.