We documented the fate of 29 cohorts of propagated Barrens topminnows Fundulus julisia stocked as juveniles and adults (n_total = 2770 fish) into 17 springheads and small ponds in middle Tennessee in 2003 and 2004. Annual mortality rates were calculated after estimating the number of individuals of each cohort remaining 1–18 mos after fish were stocked. Lighted larval fish traps were deployed at seven reintroduction sites and the Type Locale to determine whether topminnows could reproduce in the presence of the introduced-transplanted Western mosquitofish Gambusia affinis. At stocking sites harboring mosquitofish (n = 12), their density ranged from 0.4 to 66.3 mosquitofish per m². Annual mortality of stocked Barrens topminnows ranged from 45 to 100% and 24 cohorts experienced annual mortality greater than 95%. Mortality was not related to mosquitofish density or the mean size at stocking. The robustness of Barrens topminnows did not differ in the presence or absence of mosquitofish, suggesting that interspecific competition for food was not occurring. Larval Barrens topminnows were collected at two reintroduction sites and the Type Locale, but juvenile recruits were produced only at sites lacking mosquitofish. The findings of this study, and concurrent laboratory studies, support the hypothesis that mosquitofish predation on larval Barrens topminnows was the primary mechanism in failed reintroductions and is the greatest threat to wild and reintroduced populations of this imperiled species.