We have generated potent Ag-nonspecific cytolytic activity in PBMC cultures without exogenous IL-2 by stimulating the cells with anti-CD3 mAb. Three anti-CD3 mAb (147, 446, 454) each induced cytolytic activity against both K562 and Daudi targets, albeit to different degrees. The differences among the anti-CD3 mAb could not be explained by differences in their isotypes or avidity constants, by the number of anti-CD3 mAb molecules initially bound per cell, or by preferential or differential binding to TCR alpha/beta+ vs TCR gamma/delta+ cells. Each anti-CD3 mAb appeared to induce cytolytic activity via, in part, an IL-2-independent component, as evidenced by: 1) the generation of potent cytolytic activity in mAb 446-stimulated cultures despite undetectable IL-2 levels; 2) the frequent inability of exogenous IL-2 (10 U/ml) to generate as much cytolytic activity as that induced by mAb 147 or 454, despite the low levels of IL-2 (less than U/ml) in the latter cultures; and 3) the different kinetics in generation of cytolytic activity between IL-2 and anti-CD3 mAb. Moreover, exogenous IL-2 enhanced anti-CD3-induced cytolytic activity for each anti-CD3 mAb. However, IL-2-dependent processes also contributed to the generation of cytolytic activity, because anti-p55 IL-2R mAb in combination with anti-p75 IL-2R mAb partially inhibited generation of anti-CD3-induced cytolytic activity, albeit to a lesser degree than the inhibition by these anti-IL-2R mAb of generation of IL-2-induced cytolytic activity. By demonstrating the generation of potent cytolytic activity in the absence of exogenous IL-2, these studies may assist in the development of more effective and less toxic clinical adoptive immunotherapy protocols.