In two Messor species, M. meridionalis and M. foreli, glandular source, optimum concentration, longevity and interspecific responses of the trail pheromone were studied in the laboratory. Dufour's gland is the sole origin of the trail pheromone, and has no synergism with the content of the poison gland or the hindgut. Secretions of other gaster complex glands, as well as hindgut contents, in fact, did not evoke trail following. The optimum concentration of trail pheromone was found to be one gland equivalent/30 cm trail. This concentration demonstrated effective longevity for about 1 h. Interspecific trail-following tests indicate that signals mediating trail-following behaviour in these harvesting ants are not strictly species-specific.