We have monoclonal antibodies (mAbs) against CD4-1 (6D1) and CD8α (2C3) in ginbuna crucian carp Carassius auratus langsdorfii. In our previous studies we showed that 2C3 mAb positive cells are the primary cell type showing specific cytotoxicity against allogeneic targets, suggesting that CD8α+ lymphocytes in ginbuna are equivalent to cytotoxic T lymphocytes (CTLs) in mammals. We further demonstrated the helper T cell function of 6D1 mAb positive cells by studying mixed leukocyte culture (MLC) and hapten/carrier effects. Here, we report that our mAbs cross-react with zebrafish lymphocytes. First, mAbs 6D1 and 2C3 recognized 7–11% of zebrafish lymphocytes that were ZAP-70 positive and had the typical morphology of lymphocytes. Second, to verify the cell types reacting with the 6D1 and 2C3 mAbs we examined the expression profiles of zebrafish lymphocyte surface markers in FACS-sorted lymphocytes from kidney. cd4-1 (cd8a) and tcrac but not iglc transcripts were detected in 6D1(2C3)+ lymphocytes, whereas cd4-1 (cd8a) transcripts were not found in 6D1 (2C3)- lymphocytes. Third, we further confirmed that 6D1 reacted with zebrafish CD4-1 but not CD4-2, and 2C3 recognized zebrafish CD8α expressed on HEK293T cells. Collectively, these findings suggest that 6D1+ and 2C3+ lymphocytes in zebrafish are equivalent to CD4+ and CD8α+ T lymphocytes in mammals, respectively. Furthermore, we found the cross-reactivity of our 6D1 and 2C3 mAbs with other cyprinid species including goldfish, common carp and grass carp.