ABSTRACT: Analysis of the lipid composition of gonads allows differentiation between energy and structural lipids, which is important for understanding lipid utilization during gametogenesis. Fatty acids, which are frequently used as biomarkers in trophic ecology studies, are also a useful companion to lipid studies and provide a detailed understanding of a species’ reproductive cycle. We examined the influence of sex and the reproductive cycle on the lipid and fatty acid profiles of gonads in the sea urchin Arbacia dufresnii. In a population from Nuevo Gulf, Argentina, we found significant changes in total lipid concentration, lipid profile and fatty acid profile throughout the reproductive cycle. Ovaries and testes containing fully mature gametes differed in total lipid concentration (higher in the ovaries), lipid profile (more energy lipids in ovaries and more structural lipids in testes) and fatty acid profile (differed by sex, not only in the variety of fatty acids present but in the degree of unsaturation). Our results show that differences in lipid and fatty acid profiles caused by sex and gonadal reproductive stage need to be considered when these tools are used as biomarkers in ecological studies.