In Dunaliella salina, we studied the early steps in the isoprenoid pathway for the biosynthesis of carotenoids and beta-carotene and the effects of metabolic inhibitors. When D. salina was grown under carotenogenic and non-carotenogenic conditions, mevinolin did not inhibit growth or the accumulation of carotenoids, beta-carotene or chlorophyll. In contrast, fosmidomycin progressively inhibited cell growth and the biosynthesis of carotenoids, beta-carotene and chlorophyll. In this work, we reported for the first time that in D. salina, beta-carotene biosynthesis does not proceed via the classical acetate/mevalonate pathway but via the novel glyceraldehyde 3-phosphate/pyruvate pathway. This favours the yield of C(5) isoprenoid units for synthesis of isopentenyl diphosphate, the precursor in the biosynthesis of C(20) compounds, including geranylgeranyl diphosphate. Consequently, this pathway promotes carotenogenesis and the biosynthesis of C(40) beta-carotene in D. salina.