Abstract: The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1?, cds1, and ino2? mutant cells which produce 'supersized' LDs. LDs of fld1? and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2? strains had a higher (cds1) or lower (ino2?) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2?, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2? strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome.