To explore the relationship between anti-interferon-alpha (anti-IFN-alpha) antibodies and loss of clinical responsiveness to IFN-alpha treatment, we examined sera from 59 patients with hairy cell leukemia who responded to therapy with recombinant IFN-alpha-2a (rIFN-alpha-2a). During the first 2 years of therapy, 10 patients developed rIFN-alpha-2a-neutralizing and 15 rIFN-alpha-2a-binding antibodies. Nine of the 59 initially responding patients became resistant to rIFN-alpha-2a and suffered a relapse of the disease at 7 to 24 months of treatment. All nine relapsing patients tested positive for both neutralizing and binding antibodies with titers above 400 INU/mL, while none of the antibody-negative patients relapsed. Six patients with detectable binding antibody titers below 400 INU/mL continued to respond to treatment. By measuring the IFN kinetics and the levels of the IFN-induced Mx-homologous protein in mononuclear cells after a single injection each of rIFN-alpha-2a and nIFN-alpha the IFN antibodies of eight of the nine resistant rIFN-alpha patients were found to be highly specific for rIFN-alpha-2a. Therefore, these eight patients were switched to natural IFN-alpha (nIFN-alpha) therapy at doses of 3 million IU, three times a week. All eight patients responded to treatment with nIFN-alpha, achieving durable objective responses similar to those obtained previously with rIFN-alpha-2a. These data clearly demonstrate that rIFN-alpha antibody-positive patients can effectively be treated with nIFN-alpha.