Experiments and theory in single trophic level systems dominate biodiversity and ecosystem functioning research and recent debates. All natural ecosystems contain communities with multiple trophic levels, however, and this can have important effects on ecosystem structure and functioning. Furthermore, many experiments compare assembled communities, rather than examining loss of species directly. We identify three questions around which to organise an investigation of how species loss affects the structure and functioning of multitrophic systems. 1) What is the distribution of species richness among trophic levels; 2) from which trophic levels are species most often lost; and 3) does loss of species from different trophic levels influence ecosystem functioning differently? Our analyses show that: 1) Relatively few high-quality data are available concerning the distribution of species richness among trophic levels. A new data-set provides evidence of a decrease in species richness as trophic height increases. 2) Multiple lines of evidence indicate that species are lost from higher trophic levels more frequently than lower trophic levels. 3) A theoretical model suggests that both the structure of food webs (occurrence of omnivory and the distribution of species richness among trophic levels) and the trophic level from which species are lost determines the impact of species loss on ecosystem functioning, which can even vary in the sign of the effect. These results indicate that, at least for aquatic systems, models of single trophic level ecosystems are insufficient for understanding the functional consequences of extinctions. Knowledge is required of food web structure, which species are likely to be lost, and also whether cascading extinctions will occur.