Microalgae have high photosynthetic efficiencies and produce many valuable compounds from carbon dioxide. The Dunaliella genus accumulates glycerol, yet no commercial process currently exists for glycerol production from this microalga. Here it was found that in addition to intracellular accumulation, Dunaliella tertiolecta also releases glycerol into the external medium continuously, forming a large and stable carbon pool. The process is not affected by nutrient starvation or onset of cell death. Carbon dioxide was fixed at a constant rate, the bulk of it being channelled to extracellular glycerol (82%), resulting in enhanced photosynthetic carbon assimilation of 5 times that used for biomass production. The final extracellular glycerol concentration was 34 times the maximum concentration of intracellular glycerol; the latter declined further during cell death. Findings from this work will assist in the development of a bioconversion process to produce glycerol using D. tertiolecta without the need for cell harvest or disruption. Crown Copyright (C) 2013 Published by Elsevier Ltd. All rights reserved.