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, influencing 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 evalúate the effect of granulated rockwool substrate (Agrolan , Compañía IndustrialEl 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-modified hydroponic system. Growth analyses were performed at the 2 to 3 trae leaf stage of the lettuce seedlings, which were subsequently transferred to an NFT-modified hydroponic system. The stress level was evaluated at 10 days after transplant. From emergence until the 2 to 3 trae leaf stage, no significant 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 significant (p ≤ 0.05) for all evaluated parameters 10 days after transplanting. These results confirmed 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-modified hydroponic system.