Use of the monoclonal antibody OKT3 to prevent or treat allograft rejection has become commonplace. Its administration is often complicated by serious side effects, usually occurring within one to two hours after OKT3 is given, and is termed first-dose reaction. The mechanism underlying these signs and symptoms is poorly defined, but may be related to cytokine release. Twenty-three kidney or kidney/ pancreas transplant recipients received OKT3 as treatment of acute rejection. Signs and symptoms occurring after the first dose were observed and quantitated prospectively, and a reaction score was calculated. Blood was drawn immediately before, and at 2 and 24 hours after the first dose of OKT3 for determination of interleukin-2 (IL2), interferon-gamma (IFNγ), and tumor necrosis factor-alpha (TNFα) levels and flow cytometric analysis of T cell subsets. Two groups were defined based on severity of first-dose reaction. Group 1 patients (N = 11) had very mild reactions (reaction score ≤ 3); Group 2 patients (N = 12) had more severe reactions (score ≥ 5). All patients demonstrated a significant rise in serum TNFα from baseline to two hours after OKT3 (9 ± 3 pg/ml to 378 ± 54 pg/ml, P < 0.0001), and there was significant correlation between reaction scores and two-hour TNFα levels (P = 0.005). Group 2 patients had higher TNFα levels at two hours than did Group 1 patients (484 ± 75 pg/ml vs. 263 ± 62 pg/ml, P = 0.04). Levels of IL2 and IFNγ were not elevated at any sampling time. Group 2 patients also had significantly greater numbers of circulating CD3+ (521 ± 114 vs 257 ± 58, P = 0.04) and CD4+ (226 ± 45 vs. 87 ± 23, P = 0.01) lymphocytes prior to OKT3 administration. In addition, pre-OKT3 CD4+ levels correlated with reaction scores. These data implicate TNFα, perhaps of activated T lymphocyte origin, as a key mediator of the OKT3 first-dose reaction.