Partial purification of a sperm maturation factor (SMF) in the intertidal polychaete Arenicola marina has implicated arachidonic acid, an arachidonic metabolite, or a similar substance as the active factor from the prostomium. The effects of a number of 20-carbon fatty acids on inactive spermatozoa are investigated, and this reveals that only arachidonic acid and 8,11,14-eicosatrienoic acid cause sperm activation. The use of argentation thin-layer chromatography to separate fatty acids with varying degrees of unsaturation reveals a component in prostomial lipid extract, which co-migrates with eicosatrienoic acids. Investigations using cyclooxygenase and lipoxygenase result in a loss of sperm-activating properties of both prostomial extract and fatty acids. The use of cyclooxygenase and lipoxygenase inhibitors has no effect. Bovine serum albumin (BSA) reduces the sperm activating properties of both fatty acids and prostomial extract in a dose-dependant way. Additional purification procedures using: (a) organic solvents and aqueous buffers and (b) ODS silica cartridges, demonstrate that the active fraction of prostomial extract co-elutes at every step with the 8,11,14-eicosatrienoic acid standard. Gas chromatography of methyl esters of prostomial lipid extract reveals the presence of a peak with an identical retention time to the methyl ester of authentic 8,11,14-eicosatrienoic acid standard. The results described here provide strong evidence that the active SMF in prostomial homogenate is not a fatty acid metabolite but the parent acid 8,11,14-eicosatrienoic acid. These results could only be made unequivocal by full structural analysis using mass spectrometry and NMR following capillary gas-liquid chromatography.