The use of a panel of monoclonal antibodies (mAb) raised against recombinant (serine-17) human interferon-beta (rHuIFN-beta ser) has permitted the identification of three epitopes on HuIFN-beta, designated as sites I, II and III, based solely on functional differences, i.e., the neutralization of antiviral and antiproliferative activities of natural and recombinant HuIFN-beta (Redlich, P.N. and Grossberg, S. E., J. Immunol. 1989. 143: 1887). Site I- and II-directed mAb possessed neutralizing activity whereas none was noted by mAb recognizing site III. To characterize further these epitopes by immunochemical means, we studied their (a) spatial relationship by competitive binding assays, (b) antigenic structure by Western blotting, and (c) sensitivity to chemical modification by the measurement of mAb reactivity after radioiodination. Competitive antibody binding studies revealed site II to be spatially distinct from sites I and III. Furthermore, site I- and II-directed mAb could easily recognize rHuIFN-beta ser on a Western blot, suggesting that both these epitopes are primarily sequential in structure or denaturation resistant. Chemical modification by radioiodination, which did not alter the biologic activity of rHuIFN-beta ser, had likewise little effect on mAb reactivity to site I; however, reactivity to site II was diminished and reactivity to site III was minimal following the radioiodination process. Both site I- and II-directed mAb inhibited the binding of 125I-rHuIFN-beta ser to intact Daudi cells, suggesting that inhibition of receptor binding is their mechanism of neutralization. Thus, we conclude that epitopes I and II, which are associated with both antiviral and antiproliferative activities of rHuIFN-beta, are spatially and immunochemically distinct.