The Colorado River below Lake Mead, which supplies drinking water for approximately 20,000,000 people, is contaminated by ammonium perchlorate. We identified populations who were exposed and unexposed to perchlorate-contaminated drinking water and compared median newborn thyroid-stimulating hormone (TSH) levels after adjusting for age in days at measurement and for race/ethnicity. Median newborn TSH levels in a city whose drinking water supply was 100% perchlorate-contaminated water from the Colorado River below Lake Mead were significantly higher than those in a city totally supplied with non–perchlorate-contaminated drinking water, even after adjusting for factors known or suspected to elevate newborn TSH levels. This ecological study demonstrates a statistically significant association between perchlorate exposure and newborn TSH levels. It suggests that even low-level perchlorate contamination of drinking water may be associated with adverse health effects in neonates and highlights the need for both further study and control of human low-level perchlorate exposure.