The quality of lettuce (Lactuca sativa) seedlings is closely dependent on the substrate and the management practice during the transplant to the hydroponic system. This seed bed process may affect the normal development of the seedlings by affecting the post-transplanting period, potentially causing root stress, infl uencing establishment and even leading to seedling loss. An important criterion when choosing either a single or mixed substrate is the ability to provide for both growth and establishment of the seedlings in the hydroponic system. The main objective of this study was to evaluate the effect of granulated rockwool substrate (Agrolan™, Compañía Industrial El Volcán S.A., Santiago, Chile), expanded perlite A6 (Harbolite Chile Ltda., Santiago, Chile) and a mixture of both substrates on seedling growth, development and establishment after transplant of lettuce seedlings to an NFT-modifi ed hydroponic system. Growth analyses were performed at the 2 to 3 true leaf stage of the lettuce seedlings, which were subsequently transferred to an NFT-modifi ed hydroponic system. The stress level was evaluated at 10 days after transplant. From emergence until the 2 to 3 true leaf stage, no signifi cant differences were observed between the assayed substrates. However, there was a consistent tendency for better growth development when seedlings were cultivated in the 100% rockwool substrate or in the substrate mixture containing rockwool in comparison to 100% expanded perlite. The differences obtained at seedling stages were maintained after seedlings were transplanted. The effect of the substrate was statistically signifi cant (p ≤ 0.05) for all evaluated parameters 10 days after transplanting. These results confi rmed that the rockwool substrate, alone or in combination with expanded perlite, allows for the production of high-quality lettuce seedlings that optimally respond to their establishment in an NFT-modifi ed hydroponic system.