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Preventing the spread of invasive fishes is an important aspect of management programs, but is challenging due to the behavior of fish and the nature of aquatic environments. The use of dissolved carbon dioxide (CO
2
) has recently gained traction as a non-physical barrier for invasive fishes due to its ability to elicit avoidance behaviors in fish. Research to date has focused on the development of CO
2
barriers using static water environments. Because CO
2
barriers have been proposed for flowing water (i.e., in rivers or shipping canals), understanding the dynamics between fish and elevated CO
2
in flowing water is essential. Our study aims to define threshold levels required to alter behavior of bluegill (
Lepomis macrochirus
) and largemouth bass (
Micropterus salmoides
) in flowing water, and to quantify behavioral metrics of fish exposed to < 200 [ambient], 25,000, 50,000, and 100,000 µatm
p
CO
2
. We also sought to quantify the impacts of repeated CO
2
exposure on fish behavior. Bluegill showed increased activity at 25,000 µatm, while largemouth bass showed increased activity at 100,000 µatm. When repeatedly exposed to cycles of 50,000 µatm
p
CO
2
, bluegill exhibited increased activity followed by a diminished response after the second exposure. Results from this study define threshold levels required to elicit behavioral responses, and show that the effects that multiple exposures of elevated
p
CO
2
can decline, possibly due to habituation. Results will help shape the development and deployment of a CO
2
barrier to control the movements of invasive fishes.