The fatty acid compositions were determined for total lipids, triacylglycerols, phospholipids and four phospholipid fractions, including phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine/phosphatidylinositol (PS/PI) and cardiolipin (CA) obtained from hemocytes and cell-free serum from second day, fifth instar larvae of the tobacco hornworm Manduca sexta and the standard Manduca rearing medium. The hemocyte fatty acid profiles were considerably different from the profiles of the medium the insects were reared on and from the profiles of the cell-free serum. Hemocyte neutral lipids had lower proportions of polyunsaturated fatty acids than phospholipids. The fatty acid profiles of PC, PE, PS/PI and CA differ from each other and from the total lipid profiles, indicating selective fatty acid incorporation into hemocyte phospholipid species. Studies with radioactive arachidonic acid similarly indicated selective incorporation of polyunsaturated fatty acids into hemocyte lipids. Under our in vitro conditions, > 40% of the total radioactivity was incorporated into hemocyte lipids. About 93% of the incorporated radioactivity was found in phospholipids. Within phospholipids. most of the radioactivity was associated with PC (46%), and less with PE (28%) and PS/PI (21%). Very little radioactivity was recovered in CA (0.9%).