Fatty acid signature analysis (FASA) makes use of specific fatty acids, as well as entire profiles, to study dietary relationships at different trophic levels. Previously, FASA has been used in marine ecosystems in which diet determination by more direct methods is difficult and sometimes misleading. This study examined fatty acid profiles in milk from 2 species of pinniped from the Southern Ocean that were expected to have highly contrasting diets. Milk samples were collected from Antarctic fur seals Arctocephalus gazella in 3 consecutive years, from 1991 to 1993 (n = 72), and from Southern elephant seals Mirounga leonina in 1988 (n = 53) at South Georgia. Lipids were extracted and fatty acid profiles determined by temperature-programmed gas chromatography. Possible prey species collected from waters around South Georgia were also analysed. Cluster analysis as well as classification and regression trees (CART) indicated that profiles from fur seals and elephant seals were significantly different. Southern elephant seal data could be distinguished from Antarctic fur seals by lower levels of the fatty acids 16:4 n1, 18:2 n6, 18:4 n3, 18:4 n1 and 20:5 n3 and by higher levels of 18:0, 18:1 n9/ n11 (i.e. 18:1 n9 co-eluting with 18:1 n11) and 20:1 n9. Fatty acid signatures from the milk of Antarctic fur seals were closest to krill and fish species that were also known to feed on krill. Southern elephant seal fatty acid profiles were closest to species that are not known as krill predators such as larger notothenids and myctophids. The fatty acid profiles of Antarctic fur seals showed considerable inter- and intra-annual variability, which was congruent with diet variability detected using scat analyses. Southern elephant seals showed little variation in profile through lactation. In contrast to previous diet analyses based on examination of stomach contents, the results from FASA were consistent with a fish-based diet for Southern elephant seals.