- The halotolerant microalgae Dunaliella bardawil accumulates under nitrogen deprivation two types of lipid droplets: plastoglobuli rich in β-carotene (βC-plastoglobuli) and cytoplasmatic lipid droplets (CLD). We describe the isolation, composition and origin of these lipid droplets. Plastoglobuli contain β-carotene, phytoene and galactolipids, missing in CLD. The two preparations contain different lipid-associated proteins: major lipid droplet protein (MLDP) in CLD, and the proline-rich carotene globule protein (CGP) in βC-plastoglobuli. The compositions of triglycerides (TAG) molecular species, total fatty acids and sn-1+3 and sn-2 positions in the two lipid pools are similar, except for a small increase in palmitic acid in plastoglobuli, suggesting a common origin. The formation of CLD TAG precedes that of βC-plastoglobuli, reaching a maximum after 48h of N deprivation and then decreasing. Palmitic acid incorporation kinetics indicated that at early stages of N deprivation CLD TAG is synthesized mostly from newly formed fatty acids, whereas in βC-plastoglobuli large part of TAG is produced from fatty acids of pre-formed membrane lipids. Electron microscopic analyses revealed that CLD adhere to chloroplast envelope membranes concomitant with appearance of small βC- plastoglobuli within the chloroplast. Based on these results we propose that CLD in D. bardawil are produced in the endoplasmatic reticulum (ER) whereas βC-plastoglobuli are made in part from hydrolysis of chloroplast membrane lipids and in part by a continual transfer of TAG or of fatty acids derived from CLD.