The lipid composition of platelet cytoskeletons was analyzed. Triton X- 100 (0.5%) was used to prepare cytoskeletons from thrombin-treated platelets. The lipid/protein ratio of platelet cytoskeletons was 0.260 and the phospholipid/protein ratio was 0.177, which were comparable to the ratios present in platelets. However, there was a selective enrichment of platelet lipids in platelet cytoskeletons. Only 2 of the 5 major platelet phospholipids were detected. About 14% platelet sphingomyelin and 2% platelet phosphatidylcholine were present in platelet cytoskeletons. Only 1 of the 4 platelet neutral glycolipids, trihexosyl ceramide, was detected and was about 7% of that in intact platelets. Two percent of platelet hematoside, the predominant ganglioside in platelets, was found in cytoskeletons. Six percent of platelet cholesterol was present in platelet cytoskeletons, while no other neutral lipid could be detected. The study demonstrates that the lipid/protein ratio of platelet cytoskeletons is similar to that in platelets, but the composition of cytoskeleton lipids is specific and distinctly different from that in platelets. The selective glycolipid and phospholipid composition of cytoskeletons may be important for cytoskeleton and platelet function.