Sexual isolation between two species of North American plethodontid salamanders (Desmognathus imitator and D. ochrophaeus) from a locality where they are sympatric was investigated by staging a series of 40 intraspecific and 40 interspecific sexual encounters. Only 5% of interspecific encounters resulted in insemination of the female, indicating a high degree of sexual isolation between these species (Stalker's index of sexual isolation = + 0.88). Behavioral observation revealed that, in 80% of interspecific sexual encounters, males apparently ignored heterospecific females and failed to initiate even the preliminary stage of courtship, during which the male actively pursues the female. Evidence of a role for chemical stimuli in maintaining sexual isolation was obtained in experiments in which males were exposed to different types of odors in a Y-maze olfactometer. Males of both D. imitator and D. ochrophaeus preferred conspecific over heterospecific female odor. This result supports the hypothesis that interspecific courtships fail because heterospecific females produce inappropriate chemical cues.