Thiol modifiers and oxidants inhibit lymphocyte activation. To investigate which of the many cell functions sensitive to oxidation are critical in this inhibition, mouse splenic lymphocytes were treated with oxidants prior to exposure to mitogen, and progression into the cell cycle was assayed. Different treatments were used to chemically dissect different potential targets within the cell: copper phenanthroline (CuP), to oxidize surface sulfhydryls; N-ethyl maleimide (NEM), to alkylate extra- and intracellular thiols; and hydrogen peroxide, which generates the highly reactive hydroxyl radical within the cell. Progression into the cell cycle was assayed with acridine orange (AO) and assays of interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. The contribution of ADP-ribosylation to inhibition of mitogenesis was assessed using 3-aminobenzamide (3AB) to inhibit adenosine 5'-diphosphate (ADP)-ribose transferases. The results indicate that the CuP and NEM treatments both produce two independent inhibitory effects, that is, a failure in the production of and response to IL-2. Cells treated with these compounds were able to progress only through G1a upon mitogenic stimulation. H2O2 had more complex effects. Both ADP-ribosylation and modulations of cytosolic Ca2+ were involved in the inhibitory effects. With lower inhibitory doses of H2O2, lymphocytes were completely unresponsive to mitogen and failed to exit Go upon mitogenic stimulation. If intra- and extracellular Ca2+ were buffered before treatment with H2O2, higher concentrations were required, and under these conditions cells were able to enter G1a but could not progress into G1b. Under neither of these conditions could cells produce IL-2 or express IL-2R.