High levels of ether phospholipids were found in rat platelets. Alkylacyl compounds constituted 18 and 29% of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). Alkenylacyl compounds, not detected in GPC, represented 40% of GPE. Arachidonate comprised 60%, 42% and 26% of the acyl residues in the sn-2 position of alkenylacyl-GPE, alkylacyl-GPE and alkylacyl-GPC respectively. Based on all arachidonate being linked to the sn-2 position of the diacyl species, the arachidonate level was 47% in diacyl-GPE and 30% in diacyl-GPC. The incorporation and metabolic fate of arachidonate in various phospholipid classes of resting platelets was examined. Arachidonate was essentially recovered in the diacyl phospholipids and very poorly in alkylacyl- and alkenylacyl-GPE and GPC after 30 min incubation in the presence of [14C]arachidonic acid. Upon reincubation of the platelets after removal of free arachidonate, the radioactivity was gradually lost from diacyl-GPC. Concomitantly, the radioactivities in alkylacyl-GPC, alkylacyl-GPE, alkenylacyl-GPE and to a lower extent in diacyl-GPE were increased. Labelling of glycerophosphoinositol was not changed. This labelling transfer was linear up to 5-6 h, except for alkylacyl-GPC; then labelling remained constant. These data strongly suggest that free arachidonate incorporation through the Lands pathway occurs only for diacyl species and that arachidonate incorporation into the ether phospholipids is achieved by exchange from diacyl-GPC. Based on specific activities related to phosphorus content, the arachidonate incorporation rates into diacyl-GPE and diacyl-GPC were approximately equivalent. The very large differences between specific radioactivities related to arachidonate observed at the starting reincubation time were strongly attenuated when labelling equilibrium was reached. The turnover rate by this exchange pathway was higher in alkylacyl-GPC than in alkyl- and alkenylacyl-GPE. This finding agrees with the selectivity for arachidonate observed in the acylation of PAF-acether in human neutrophils [Chilton, O'Flaherty, Ellis, Swendsen & Wykle (1983) J. Biol. Chem. 258, 7268-7271].