The mating behaviour of the signal crayfish, Pacifastacus leniusculus (Dana), has been categorised into seven distinct stages:; orientation; contact; seizure; turning; mounting; spermatophore deposition; and dismounting. These categories were used in the development of a bioassay to test whether mature females release a sex pheromone during the breeding season to which males respond. Water conditioned by 1) mature females (MF water), 2) immature females (IF water) and 3) freshwater (C water), were injected through standard aquarium air-stones into tanks containing mature males. The behaviour of the male was recorded on video for 15 minutes before and after injection of each test water. The time that the males spent exhibiting the three behavioural categories of quiescent, motile and handling of the air-stone, were recorded. Handling of the air-stone included the mating behaviours of seizure, mounting, and spermatophore deposition onto the surface of the air-stone. Males exposed to MF water exhibited significantly increased levels of motile activity and handling behaviour, than males exposed to C water or IF water. These results demonstrate for the first time the presence of a sex pheromone, released during the breeding season by mature females, that stimulates courtship and mating behaviour in male P. leniusculus.