Gaps remain in our knowledge about the levels of fluoride intake that cause dental fluorosis. The purpose of this study was to report the fluorosis prevalence by levels of estimated fluoride intake in an effort to understand the importance of different levels of daily fluoride intake. As part of the longitudinal Iowa Fluoride Study, subjects were followed from birth to 36 months with questionnaires every 3-4 months to gather information on fluoride intake from various sources. Daily fluoride intake in mg per kg body weight (BW) was estimated from water, beverages and selected foods, fluoride supplements and dentifrice. Six hundred and twenty-eight subjects were examined for fluorosis on permanent incisors and first molars at about age 9 by two calibrated examiners using the Fluorosis Risk Index categories. Fluorosis prevalence rates were determined separately for maxillary central incisors and first molars by levels of estimated fluoride intake. There were significant positive associations between fluorosis prevalence and levels of fluoride intake. Cumulatively from birth to 36 months, average daily intake of 0.04 mg F/kg BW or less carried relatively low risk for fluorosis (12.9% for maxillary central incisors, 6.8% for first molars). Average daily intake of 0.04-0.06 mg F/kg BW showed a significantly elevated risk for fluorosis (23.0% for maxillary central incisors, 14.5% for first molars), while fluorosis risk was even higher for average intake above 0.06 mg F/kg BW (38.0% for maxillary central incisors, 32.4% for first molars). The study suggests that fluorosis prevalence is related to elevated fluoride intake when averaged over the first 3 years of life, but is even more strongly related to fluoride intake that is elevated for all of the first 3 years of life.