Epstein-Barr virus (EBV)-specific T cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Whilst these preparations are enriched for EBV antigen-specific CD8(+) T cells, most also contain a CD4(+) T cell population whose specificity is unknown. Here we show that, although CD4(+) T cell clones derived from such cultures recognise HLA class II-matched LCLs but not mitogen-activated B lymphoblasts, many (i) do not map to any known EBV antigen, (ii) can be raised from EBV-naive as well as EBV-immune individuals, and (iii) can recognise a broad range of human B-lymphoma-derived cell lines irrespective of EBV genome status, providing those lines express the relevant HLA class II restricting allele. Importantly, such CD4(+) clones not only produce IFNgamma, but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in co-cultivation assays. We infer that such CD4(+) T cells recognise cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B cell malignancies. Such antigens are therefore of potential value as targets for CD4(+) T cell-based immunotherapy.