To examine the C 20 polyunsaturated fatty acid (PUFA) compositions of Collembola, we raised five species of Collembola on yeast diets, and then quantified body mass, neutral lipid fatty acid (NLFA) and phospholipid fatty acid (PLFA) compositions. PLFA content was always less than 5% of dry weight, but NLFA content varied from 5.9% to 29.6% of dry weight, depending upon species. Combined C 20 PUFA proportions of up to 9.2% and 48% were observed in the NLFA and PLFA fractions, respectively, resulting in total C 20 PUFA proportions of up to 19.4% of the total fatty acid compositions of Collembola. C 20 PUFAs were also detected in Collembola specimens from a deciduous woodland at proportions up to 29.7% of the total fatty acid composition. Terrestrial invertebrates generally contain <4% and <22% C 20 PUFAs in PLFAs and NLFAs, respectively; therefore, these results demonstrate that Collembola often possess the highest proportions of C 20 PUFAs yet observed in terrestrial invertebrates. The biochemical reasons for such high C 20 PUFA proportions, which were biosynthesised by the Collembola since these components were absent from the yeast diets, remain unclear. The distinctive fatty acid compositions of Collembola may be useful in soil food web studies utilising fatty acids as biomarkers of trophic behaviour.