Etheostoma zonale, the banded darter, was introduced to the Susquehanna River basin, Pennsylvania, through an interbasin transfer. We examined the effects of darter density and the presence of E. zonale on the substrate choice of two native darter species (Etheostoma olmstedi, the tessellated darter, and Percina peltata, the shield darter) in an artificial stream. In single species trials, E. olmstedi, E. zonale, and P. peltata exhibited nonrandom substrate selection and occupied patches of large substrate significantly (P < 0.05) more often than expected. No intraspecific density effects were observed. The presence of E. zonale did not affect the substrate choice of P. peltata but did induce a shift of E. olmstedi from large to small substrate in high-density trials (P < 0.05). Etheostoma zonale was most frequently the aggressor in behavioral interactions. Neither E. olmstedi nor P. peltata directed any agonistic behavior toward E. zonale; however 40% of aggressive acts initiated by E. zonale were directed toward E. olmstedi and P. peltata. Displacement of E. olmstedi by the aggressor E. zonale suggests potential deleterious effects on the native species.