SynopsisAggression by nest-guarding male johnny darters, Etheostoma n&urn, against intruding crayfish was investigated in laboratoy experiments and field observations. In the laboratory, darter success in chasing crayfish, Orconectes rusticus, from the nest site was inversely related to crayfish size. Small crayfish (less than 15 mm carapace length) were routinely evicted from the nest area by nips directed at the posterior end of the abdomen. Although such aggressive behavior was less successful against larger crayfish, even the largest crayfish tested (carapace length 30-32 mm) were chased from the nest area in 33% of the trials. Those large crayfish that entered nests often remained despite repeated attacks by the male johnny darter and egg predation was observed. In a small Ohio stream, openings to johnny darter nests were generally between 7 and 13 mm. Thus crayfish with a carapace height greater than 13 mm (corresponding to a carapace length greater than 29mm) would not be able to enter johnny darter nests. In field observations, male johnny darters successfully defended nests against another crayfish species (Orconectes sanborni, carapace length 12-29mm). Together, aggressive behavior and small size of nest entrances allow the johnny darter to successfully reproduce in areas with abundant crayfish.