Post-copulatory behaviour in barnacles involves a violent rocking movement of the opercular valves, which is thought to contribute to the expulsion of oocytes through the oviduct into the mantle cavity where they are fertilised. We demonstrate in this study that the seminal vesicles/testis of the subtidal barnacle Balanus balanus produce a biologically active factor, barnacle muscle stimulatory factor (BMSF), which causes a significant increase in cirral and body muscular activity. BMSF was identified using a combination of high performance liquid chromatography and mass spectrometry as a novel eicosanoid/oxylipin, 8,13-dihydroxyeicosapentaenoic acid. This is rapidly inactivated under mild acid conditions to form a complex range of triene and pentaene chromophore-containing products that have only been partially identified. Injection of purified BMSF into the mantle cavity of barnacles caused the rocking movements of the opercular valves as reported following fertilisation. In excised barnacles, it also caused muscular contractions of the whole body mass. The breakdown products of BMSF, however, were without such activities. The function of BMSF in facilitating fertilisation in barnacles is comparable to the role of other eicosanoids in human reproduction, reinforcing the view that these compounds have conserved activities in both invertebrates and vertebrates.