Aims: To analyse the production of different metabolites by dark-grown Euglena gracilis under conditions found to render high cell growth. Methods and Results: The combination of glutamate (5 g l(-1)), malate (2 g l(-1)) and ethanol (10 ml l(-1)) (GM + EtOH); glutamate (7.15 g l(-1)) and ethanol (10 ml l(-1)); or malate (8.16 g l(-1)), glucose (10.6 g l(-1)) and NH4Cl (1.8 g l(-1)) as carbon and nitrogen sources, promoted an increase of 5.6, 3.7 and 2.6-fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of alpha-tocopherol after 120 h identified by LC-MS was 3.7 +/- 0.2, 2.4 +/- 0.1 and 2 +/- 0.1 mg [g dry weight (DW)](-1), respectively, while in the control medium (GM) it was 0.72 +/- 0.1 mg (g DW)(-1). For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP-HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. Conclusions: Dark-grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, alpha-tocopherol and paramylon. Significance and Impact of the Study: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, alpha-tocopherol and some amino acids. The concentrations of alpha-tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio-molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.