The Neosho madtom is a small, short-lived catfish species endemic to gravel bars of the Neosho River in Kansas, Oklahoma and Missouri, U.S.A. It spawns during summer in nesting cavities excavated in gravel. Although the species has survived dam construction within the Neosho River basin, its declining numbers resulted in it being added to the federal threatened species list in 1991. To test how water flow affects the reproductive behavior of Neosho madtoms, we compared activities of male-female pairs in static versus flowing-water aquaria. Using a behavioral catalog, we recorded their behavior sequences during randomly selected 5-min nighttime periods. For males and females, Jostle and Embrace were the most performed reproductive behaviors and the Jostle-Embrace-Carousel was the most performed reproductive behavior sequence. Water flow decreased the mean frequency of occurrence, percentage of time spent and mean event duration of male Nest Building. Because Neosho madtom courtship, reproduction and parental care is a complex and extended process, disturbances such as heightened river flows during the species' spawning season may negatively affect nest quality and reproductive success.