Objective: To assess the effect of gestational perchlorate exposure through drinking water on neonatal thyroxine (T4). Design: T4 values were compared among newborns in Ramat Hasharon, Israel, whose mothers resided in suburbs where drinking water contained perchlorate ≤340 μg/L (very high exposure, n = 97), 42-94 μg/L (high exposure, n = 216), and <3 μg/L (low exposure, n = 843). In the very high and high exposure areas, T4 values in newborns whose mothers drank tap water exclusively (as determined by a telephone interview) were analyzed as a subset. Serum perchlorate levels in blood from donors residing in the area were used as proxy indicators of exposure. Main outcome: Neonatal T4 values (mean ± SD) in the very high, high, and low exposure groups were 13.9 ± 3.8, 13.9 ± 3.4, and 14.0 ± 3.5 μg/dL, respectively (p = NS). Serum perchlorate concentrations in blood from donors residing in areas corresponding to these groups were 5.99 ± 3.89, 1.19 ± 1.37, and 0.44 ± 0.55 μg/L, respectively. T4 levels of neonates with putative gestational exposure to perchlorate in drinking water were not statistically different from controls. Conclusion: This study finds no change in neonatal T4 levels despite maternal consumption of drinking water that contains perchlorate at levels in excess of the Environmental Protection Agency (EPA) drinking water equivalent level (24.5 μg/L) based on the National Research Council reference dose (RfD) [0.7 μg/(kg·day)]. Therefore the perchlorate RfD is likely to be protective of thyroid function in neonates of mothers with adequate iodide intake.