Males of the ruminant nematodeTrichostrongylus colubriformis were significantly attracted to an incubate from their females during in vitro assay. Exposure of males to sera from uninfected and infected animals or selected neurotransmitters had no effect on their subsequent responsiveness to the female's pheromone. In contrast, exposure to 1500 female-hours of pheromone decreased male responsiveness after moderate rinsing prior to bioassay. Rinsing of the males with larger volumes of Tyrode's solution prior to in vitro assay increased their subsequent response to the female's pheromone. High-performance liquid chromatography yielded a presumptive pheromone peak with a fivefold increase in biological activity. This peak was soluble in alcohols and tetrahydrofuran, based on elution from reverse-phase Sep-Pak cartridges. Stability of the peak was increased by EDTA or ascorbic acid (10 mM). Storage for six weeks in ascorbic acid at 4 °C allowed recovery of 47.3% of the original material.