The role of olfaction and diffusible pheromones in mate location behavior of sea lice, Lepeophtheirus salmonis, was assessed with Y-tube behavioral bioassays. The pheromone "emitting" animals were located in a chamber in one arm of a Y-tube arena, with artificial seawater flowing through both arms. Adult male sea lice displayed both activation and directional responses to seawater conditioned with preadult II virgin females, but were only activated by mated adult female conditioned water. Further, when males were given the choice of preadult II virgin females or mated adult females, a significant number of males chose the arm with the preadult II virgin females. Adult males showed activation responses when presented with water conditioned with adult males but were not attracted to them. When presented with adult males, preadult II virgin females showed only directional responses, but not activation responses. Preadult II virgin female conditioned water was extracted using solid-phase extraction (SPE) protocols pioneered for semiochemical isolation. Adult male sea lice showed significant directional responses to the preadult II virgin female SPE extract. Distillation under vacuum was performed on the extract to give a distillate comprising components with a molecular weight range and physical properties comparable to those of compounds utilized as volatile semiochemicals by terrestrial organisms and a residue comprising components with higher molecular weight range comparable to those utilized as involatile semiochemicals. Adult males were found to be both significantly activated and attracted to the distillate, but not to the residue. This research provides evidence that small, lipophilic organic molecules are used by sea lice as sex pheromone signals to locate a member of the opposite sex.