In the present work, we followed a microarray approach to analyze the expression of glycosylation-related genes on different cell populations obtained from mouse thymus. Among other genes, transcription of the two-domain type galectin-8 was detected both in thymocytes and thymic epithelial cells (TECs), which was confirmed by reverse transcriptase (RT)-PCR assays independently carried out on both cell populations. Two splice variants, differing solely in the presence of a nine amino acid insertion in the linker peptide region connecting the two carbohydrate recognition domains (CRDs), were identified from purified thymocytes. Expression of galectin-8 was verified at the protein level in total organ extracts by western-blots of lactosyl-Sepharose purified binders. To explore the possible biological roles of locally produced galectin-8, both splice variants were recombinantly expressed in bacteria and assayed over cultured thymocytes. In spite of their binding to all cell populations, addition of either isoform of galectin-8 to thymocyte cultures induced apoptosis only of the CD4(high)CD8(high) cells through caspases pathway activation. All of these effects were prevented by the addition of thiodigalactoside (TDG) or lactose, thus indicating that the proapoptotic activity of galectin-8 was due to the specific interaction of its CRDs with defined cell surface glycans. Together, our results demonstrate intrathymic expression of galectin-8 in mouse, and suggest an active role for this lectin in shaping the mature T cell repertoire.