The effects of a variety of metabolic and anti-tumour necrosis factor (TNF) antibodies were utilized to distinguish several different mechanisms of cytotoxicity employed by CD4+ effectors isolated from human peripheral blood lymphocytes (PBL). PBL, unseparated high buoyant density T cells and their CD4+ T cell subsets were activated with anti-CD3 monoclonal antibody (MoAb) and interleukin-2 (IL-2) for 1-5 days. CD4+ T cells activated with IL-2/anti-CD3 MoAb were cytotoxic when directed by a bispecific anti-nitrophenyl (NP)-anti-CD3 MoAb heteroconjugate against both NP-modified nucleated target cells (TC) and non-nucleated sheep red blood cells (SRBC). This CD4+ T population also lysed L929 in a TNF-alpha dependent manner. Interestingly, different mechanisms of nucleated and non-nucleated TC directed lysis by CD4+ effectors were implied by distinct patterns of sensitivity to cholera toxin (CT) and cyclosporin A (CsA). Cyclosporin A and CT inhibited CD4+ T cell directed lysis of SRBC, but not EL4. Cholera toxin, CsA or EGTA pretreatment also significantly inhibited the release of alpha-N-benzyloxycarbonyl-L-lysine-thiobenzylester (BLT)-esterase activity suggesting that degranulation of CD4+ effectors may be a critical step in their redirected lysis of SRBC. Overall, these findings suggested that activated human peripheral blood (PB) CD4+ effectors can lyse TC by at least three distinct mechanisms: (i) a CsA-sensitive directed lysis of SRBC which correlates with exocytosis and presumably occurs via membrane lesions; (ii) a CsA-insensitive directed lysis of NP-modified nucleated TC that does not appear to involve exocytosis and is metabolically distinct; and (iii) a direct TNF-dependent lysis of TNF-sensitive TC. The highly proliferative CD4+ T cell population could be propagated for at least 35 days while retaining cytotoxicity and secreting up to 80 U/mL of IL-2. These data raise the possibility that anti-CD3 MoAb plus IL-2 activated CD4+ T cells may prove effective in adoptive tumour immunotherapy.