In an adjuvant clinical trial, 34 high-risk malignant melanoma patients were treated with natural interferon (IFN)-beta and recombinant IFN-gamma. Patients with tumor location on head, neck, and trunk received 3 million IU IFN-beta intravenously (IV) three times weekly for 24 weeks. Patients with tumor location on the extremities received subcutaneous (SC) injection of 2 million IU of IFN-beta distal the locoregional lymph nodes instead. All patients were given 50 micrograms IFN-gamma SC on 3 consecutive days every 4 weeks. Antibody formation was detected by coincubation of IFN and patients' serum and assessment of the inhibition of the cytopathic effect by a virus suspension. Soluble interleukin-2 receptors (sIL-2R) were determined by enzyme-linked immunosorbent assay (ELISA) technique. No antibodies against IFN-gamma were observed. The overall incidence of antibody formation to IFN-beta was 55.8% (19/34). Ninety-two percent of the SC-treated patients (13/14) and 30% (six of 20) of the IV-treated patients developed antibodies. Soluble interleukin-2 receptors were found to be significantly lower in antibody-positive patients than in antibody-negative patients. The authors conclude that IFN-beta antibody formation is frequent and might influence IFN induced sIL-2R elevation in vivo.