The structures of cytotoxic granules in interleukin-2-activated human killer T lymphocytes have been investigated by Raman microspectroscopy at a single cell level. The Raman spectra of granules share a common feature that lipid Raman bands are much stronger than the Raman bands due to protein, indicating that one of the main components of the granule is lipid. To analyze the lipid structures of individual granules, relationships between Raman spectra and structures have been examined for a series of triacylgycerols with varied degrees of acyl chain unsaturation. Analysis based on the relationships shows that the granulous lipid is characterized by a high content of cis C=C bond, which ranges from about 1.5 C=C bonds per acyl chain in isolated minor granules and to about 2.2 C=C bonds in clustering major granules. The highly unsaturated lipid of major cytotoxic granules is in sharp contrast to the moderately unsaturated (about one C=C bond per acyl chain) plasma membrane lipid. The large difference in lipid unsaturation between the granule and plasma membrane may have relevance to the role of granulous lipid in packaging cytotoxic proteins inside the granule and preventing them from attacking the killer lymphocyte itself.