Equine interferon-beta 1 (EqIFN-beta 1) was purified from extracts of recombinant Escherichia coli by sequential chromatography on hydroxylapatite, anion-, and cation-exchangers. The resulting protein was greater than 98% pure as determined by sodium dodecylsulfate gel electrophoresis, gel permeation HPLC, and reverse-phase HPLC. Amino-terminal amino acid sequencing revealed that essentially all molecules contained an additional amino-terminal methionine. The specific antiviral activity of EqIFN-beta 1 determined on equine dermal fibroblasts challenged with vesicular stomatitis virus (VSV) was approximately 5 X 10(8) U/mg. Less than 0.001% of this activity was observed in antiviral assays using human (A549), murine (L-M), ovine (SCP), or bovine (MDBK and BT) cells challenged with VSV or encephalomyocarditis virus. A series of monoclonal murine IgG antibodies were developed which neutralize the antiviral activity of EqIFN-beta 1. None of these antibodies nor rabbit antiserum to EqIFN-beta 1 were able to neutralize human IFN-beta; antiserum to human IFN-beta did not neutralize EqIFN-beta 1. Two of the monoclonal antibodies were used to establish a rapid one-step solid-phase enzyme immunoassay for EqIFN-beta 1.