Maintaining a functional pulmonary surfactant system at depth is critical for diving mammals to ensure that inspiration is possible upon re-emergence. The lipid and protein composition of lavage extracts from three pinniped species (California sea lion, Northern elephant seal and Ringed seal) were compared to several terrestrial species. Lavage samples were purified using a NaBr discontinuous gradient. Concentrations of phospholipid classes and molecular species were measured using electrospray ionisation mass spectrometry, cholesterol was measured using high-performance liquid chromatography, surfactant protein A (SP-A) and SP-B were measured using enzyme-linked immunosorbent assays. There were small differences in phospholipid classes, with a lower level of anionic surfactant phospholipids, PG and PI, between diving and terrestrial mammals. There were no differences in PL saturation or SP-A levels between species. PC16:0/14:0, PC16:0/16:1, PC16:0/16:0, long chain PI species and the total concentrations of alkyl-acyl species of PC and PG as a ratio of diacyl species were increased in diving mammals, whereas concentrations of PC16:0/18:1, PG16:0/16:0 and PG16:0/18:1 were decreased. Cholesterol levels were very variable between species and SP-B was very low in diving mammals. These differences may explain the very poor surface activity of pinniped surfactant that we have previously described [Miller, N.J., Daniels, C.B., Schürch, S., Schoel, W.M., Orgeig, S., 2005. The surface activity of pulmonary surfactant from diving mammals. Respir. Physiol. Neurobiol. 150 (2006) 220-232], supporting the hypothesis that pinniped surfactant has primarily an anti-adhesive function to meet the challenges of regularly collapsing lungs.