The aim of this study was to examine the tolerability, antitumor activity,and biological effects of a new schedule of i.v. recombinant human interleukin 12 (rhIL-12). Twenty-eight patients were enrolled in a Phase I trial in which rhIL-12 was administered twice weekly as an i.v. bolus for 6 weeks. Stable or responding patients were eligible to receive additional 6-week cycles until there was no evidence of disease or until tumor progression. Patient cohorts were treated with escalating doses of rhIL-12 (30–700 ng/kg). The maximum tolerated dose(MTD) was 500 ng/kg, with dose-limiting toxicities consisting of elevated hepatic transaminases and cytopenias. At the MTD(n = 14), there was one partial response occurring after 6 cycles of rhIL-12 in a patient with renal cell cancer. Two additional renal cell cancer patients treated at the MTD had prolonged disease stabilization, with one of these exhibiting tumor regression after 8 cycles of rhIL-12. IFN-γ, IL-15, and IL-18 were induced in patients treated with rhIL-12. Whereas IFN-γ and IL-15 induction were attenuated midway through the first cycle in patients with disease progression, those patients with tumor regression or prolonged disease stabilization were able to maintain IFN-γ, IL-15,and IL-18 induction. The down-modulation of IFN-γ induction during rhIL-12 treatment did not relate to IL-10 production or alterations in rhIL-12 bioavailability but was associated with an acquired defect in lymphocyte IFN-γ production in response to IL-12, IL-2, or IL-15. This defect could be partially overcome in vitro through combined stimulation with IL-12 plus IL-2. These findings show that the chronic administration of twice-weekly i.v. rhIL-12 is well-tolerated,stimulates the production of IL-12 costimulatory cytokines and IFN-γ,and can induce delayed tumor regression. Strategies aimed at maintaining IFN-γ induction, such as the addition of IL-2, may further augment the response rate to this schedule of rhIL-12.