Evolutionary theory predicts that some aquatic organisms may adapt by directional selection to limiting physical environmental conditions, yet empirical data are conflicting. We sought to test the assumption that sculpins (family Cottidae) inhabiting thermally stable springs of the southeastern United States differ in temperature tolerance and metabolism from populations inhabiting more thermally labile stream habitats. Spring populations of pygmy sculpins (Cottus pygmaeus) and Ozark sculpins (C. hypselurus) differed interspecifically in thermal tolerance from populations of stream-dwelling mottled (C. bairdi) and Tallapoosa sculpins (C. tallapoosae), and both stream and spring populations of banded sculpins (C. carolinae). No intra- or interspecific differences in thermal tolerance were found among populations of C. bairdi, C. talla poosae, or C. carolinae. Cottus pygmaeus acclimated to 15°C differed intraspecifically in routine metabolism from fish acclimated to 20° and 25°C. Cottus pygmaeus and stream-dwelling C. bairdi and C. carolinae acclimated to temperatures of 20° and 25°C showed no interspecific differences in routine metabolism. Our results suggest that some spring-adapted populations or species may be more stenothermal than stream-dwelling congeners, but a greater understanding of the interactions of other physical and biological factors is required to better explain micro- and macro habitat distributions of eastern North American sculpins.