Abstract Two murine monoclonal antibodies (mAbs) directed against different epitopes on recombinant porcine interferon-α (IFN-α) were selected and used to construct a two-site ELISA. This ELISA, when performed in a one-step version, detected about 0.5 units ml −1 of IFN-α and showed similar sensitivity but better precision than a cytopathic effect inhibition bioassay. Estimates of IFN-α in tissue culture medium by the two assays correlated well. In contrast, one or several factors in porcine serum reduced the sensitivity of the ELISA. Measurements of IFN-α in porcine serum was, however, possible in a two-step version of the ELISA, with a sensitivity of about 1 unit IFN-α ml −1. Results of ELISA and bioassay agreed, except that the ELISA possibly produced false positive results in two out of a total of 91 sera negative in the bioassay. In addition, one of 23 sera positive in the bioassay was negative in the ELISA.