The effects of the alkaline earth divalent cation Barium (Ba2+) were studied in in-vitro murine polyclonal T cell activation induced with a panel of T cell mitogens consisting of the plant mitogens concanavalin A (ConA), jacalin and phytohaemagglutinin (PHA), a mitogenic anti-Thy1 monoclonal antibody (MoAb), and an anti-murine CD3 MoAb combined with phorbol ester. All modes of T cell activation, except PHA-induced mitogenesis, were blocked in a reversible and dose-related manner by Ba2+. Blockade was evident only if Ba2+ was added within the first 6 h of stimulation, was totally reversed in a competitive fashion by addition of Ca2+ to the medium, and selectively affected interleukin 2 (IL-2) production, without interfering with expression of IL-2 receptor light chains, nor with late IL-2-dependent activated T cell growth. On the other hand, PHA-induced responses stimulated by optimal mitogen doses were resistant to the effects of Ba2+. Ba2+-resistance of PHA responses was due to IL-2-dependent activation and growth of a Ba2+-resistant T cell subset since: (i) limiting dilution analysis demonstrated that this PHA response had a much lower precursor cell frequency than control PHA responses; (ii) proliferation was blocked by anti-IL2 agents, such as cyclosporin A and anti-IL-2 receptor light chain MoAbs, which were much less effective in blocking control PHA responses. Thus, pharmacological use of Ba2+ reveals the existence of a pathway of T cell activation, induced by PHA, with differential interleukin requirements.