A specific, sensitive, and reproducible in vitro assay for antithyroid compounds and environmental goitrogens has been used to investigate antithyroid activity (AA) in small samples of water supplying 15 localities in endemic and nonendemic goiter areas of western Colombia. A significant positive correlation was observed between goiter prevalence and AA in water collected from the pipelines of these localities. Samples at the water source showed only borderline significance. No significant correlation was observed in waters between AA and total hardness (ppm) or concentrations of Ca, Mg, sulfates, chlorides, silicates, nitrates, and iodine. AA was also demonstrated by this in vitro assay in well water previously shown experimentally to be goitrogenic and that supplied the endemic goiter district of Candelaria town in western Colombia. In contrast, water from the well supplying the area of lower endemicity was found to possess little AA. These results provide experimental support for epidemiological observations that demonstrate a relationship between the sources of drinking water and goiter prevalence rates, and are consistent with previous findings indicating that organic antithyroid compounds contaminate water supplies in areas where goiter persists despite adequate iodine supplementation. (J Clin Endocrinol Metab56: 767, 1983)