Direct cytotoxicity by class II-restricted T cells has been proposed as a potential mechanism in autoimmune tissue damage, as well as in immunoregulation. We used I-A(s)-restricted non-granular cytotoxic T-cell hybridomas (BP24.29 and BP47.7), specific for self-determinants on myelin basic protein (MBP), and different monoclonal targets, in order to characterize the mechanism of killing used by these cells. An early lesion at the level of the target cell nucleus was indicated by the fact that target DNA lysis ([3H]thymidine release) proceeded 2-2.5-fold as rapidly as cytoplasmic lysis (51Cr release) over the first 14 hr after stimulation. Cytotoxicity was relatively resistant to inhibition by anti-calcium agents (TMB-8 and verapamil), even under conditions which blocked interleukin-2 (IL-2) release. Although tumour necrosis factor (TNF) has been proposed as one mediator of class II-restricted cytotoxicity, these cells (i) released no detectable TNF after stimulation with antigen, concanavalin A (Con A), or anti-CD3, (ii) readily lysed TNF-resistant targets (A20 and LS-102.9), and (iii) had no cytotoxic effect on TNF-sensitive cells (L929). Substantial 'bystander' killing of I-A-mismatched targets was observed, which was 13-37% of the cognate (I-A(s)-restricted) cytotoxicity measured in parallel. This finding may indicate an effector mechanism in autoimmune demyelination, since the myelin-forming oligodendrocytes of the central nervous system are not inducible for major histocompatibility complex (MHC) class II expression.