Patients receiving recombinant interferon-beta ser (rIFN-beta ser) for cancer and viral diseases were evaluated for formation of antibodies with IFN-neutralizing activity. The assay for serum neutralizing activity measures the ability of the sample to neutralize the antiviral activity of rIFN-beta ser. Neutralizing antibody incidence was route dependent. Of 335 patients treated intravenously, 3 were positive, representing an incidence of 0.9%. The remaining 13 antibody-positive patients had been treated subcutaneously, representing an incidence of 9.6% (13/136). In general, peak titers for neutralizing activity were low to moderate; 15 of the 16 patients (94%) had titers between 100 and 2,000 neutralizing units/ml. The incidence of neutralizing antibodies was higher in patients who had been under study for longer periods of time. Mean time of onset of neutralizing activity for the 16 positive patients was about 5 months. Because length of time on study appeared to be a possible predisposing factor for development of neutralizing antibodies, we recalculated the incidence of this activity for all patients on study for 5 months or longer (90 i.v., 71 s.c.). All of the 16 neutralizing-positive patients were on study for more than 150 days, whereas the remaining 87 in the i.v. group and 58 in the s.c. group were negative. Thus, a longer time on study appears to increase incidence, but in only a portion of subjects treated. No adverse clinical effects could be directly associated with neutralizing antibodies.