ABSTRACT The study focusses on alternative substrates with the potential to replace common substrates, such as mineral wool and perlite, as the influence of these on ecosystems and resources is being debated. To this aim, wood fibre, sheep wool and coco peat substrates were selected for testing. Leaf lettuce (Lactuca sativa L. ‘Lisboa’) is taken as the model crop for the evaluation of alternative substrates. The closed hydroponic system ebb and flow with growing in pots was used for the experiment. The parameters of the nutrient solution were EC 1.5–2 mS and pH 6–7. Biological parameters, content of nitrates, plant stress indicators, antioxidant activity (AA) as % scavenging of DPPH (AA), glutathione (GSH), ascorbate peroxidase (APX), total phenol content (TPC) and the content of chosen elements in the drain from substrates were evaluated. According to the biomass production of lettuce, the substrates can be ranked from the lowest as follows: sheep wool < wood fibre < mineral wool < perlite < coco peat. The levels of GSH and APX did not affect AA. The TPC showed the greatest effect on AA and the stress response in general. The nitrate content ranged between 426 and 686 mg · kg–1 of fresh mass. Instead of mineral wool and perlite, coco fibre could be a promising alternative organic substrate for lettuce production in the ebb and flow hydroponics system. Wood fibre and sheep wool still have unresolved circumstances regarding their physical and chemical parameters.