A laboratory behavioral assay examined intra- and interspecific responses to sex pheromone by screwworms, Cochliomyia hominivorax, and secondary screwworms, C. macellaria, in relation to the duration of colonization of C hominivorax test males. Females of C macellaria, like those of C hominivorax, were found to produce a pheromone that stimulates male copulatory attempts on contact. Newly colonized (< 22 generations) C hominivorax males did not respond to C macellaria pheromone, indicating that pheromone contributes to reproductive isolation between these two closely related species. Although long-colonized (> 200 generations) C hominivorax males did respond to C macellaria females or their extract, this behavior was infrequent and significantly less common than intraspecific responses. Depriving C macellaria adults of dietary protein did not affect the potency of female extracts, but did reduce male responsiveness to pheromone. These results provided little evidence that colonization reduces the ability of C hominivorax males to differentiate between C hominivorax and C macellaria females using sex pheromones.