Ammonia can cause adverse reproductive and mortality effects in individual fish by interacting with the central nervous system. The last published study that assessed the effects of ammonia on fathead minnow reproduction was a lifecycle study conducted in 1986. Our study's main goal was to re-evaluate ammonia toxicity on fathead minnow Pimephales promelas reproduction using a 20-day fecundity flow-through diluter method. Flow-through diluter systems have been used by regulatory agencies, such as the U.S. Environmental Protection Agency, in the past as an effective way to estimate acceptable levels of contaminants. There was a significant difference in cumulative egg production among treatments (ANOVA; F=10.167, p≤0.01, df=3). All three concentrations of ammonia tested in this study significantly reduced fecundity after 20days of exposure (Dunnett's, p≤0.05 for each treatment). The lowest un-ionized ammonia concentration (0.06mg/L at a pH of 7.3 and temperature of 25.1°C) tested during this study resulted in a 29% decrease in cumulative fecundity. Because all tested ammonia concentrations caused an effect on P. promelas reproduction, the no effect concentration was estimated to be 0.025mg/L un-ionized ammonia (2.19mg/L total ammonia–nitrogen). This estimate was determined using the U.S. EPA Toxicity Relationship Analysis Program to calculate the 10% effect concentration of ammonia on P. promelas reproduction. This value is much lower than the previous reported no effect concentration on P. promelas reproduction (0.37mg/L un-ionized ammonia or 6.43mg/L total ammonia–nitrogen) as determined from the 1986 study, which was used to determine the ammonia water quality criteria by the U.S. Environmental Protection Agency. Our results should be considered in the next revision of water quality criteria.