Nitrogen availability and light intensity affect β-carotene overproduction in the green alga Dunaliella salina. Following a previous study on high-light stress, we here report on the effect of nitrogen depletion on the growth characteristics and β-carotene as well as fatty acid metabolism of D. salina under a constant light regime in a turbidostat. Upon nitrogen depletion, the biomass yield on absorbed light approximately doubled, due to a transient increase in cell division rate, swelling of the cells and a linear increase of the density of the cells. Simultaneously, β-carotene started to accumulate up to a final intracellular concentration of 14 mg LCV⁻¹ (i.e. 2.7% of AFDW). This β-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates β-carotene in lipid globules, we also determined the fatty acid content and composition of D. salina. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation, indicating that β-carotene and total fatty acid accumulation were unrelated, similar to what was found previously for high-light treated cells. However, for both high-light and nitrogen stress, β-carotene accumulation negatively correlated with the degree of unsaturation of the total fatty acid pool and, within the individual fatty acids, correlated positively with oleic acid biosynthesis, suggesting that oleic acid may be a key component of the lipid-globule-localized triacylglycerols and thereby in β-carotene accumulation.