Human B-lymphoblastoid cell lines immortalized by Epstein-Barr virus constitutively produce low levels of acid-stable interferon (IFN) that differs in composition from IFN produced by the same cell type after virus induction. We use three approaches to study the constitutive IFN produced by the lymphoblastoid cell line, LuKII: (i) antiviral bioassays with a neutralizing monoclonal antibody; (ii) amino-terminal sequence analysis of the single protein obtained after purification of the IFN by immunoaffinity chromatography and reverse-phase HPLC; (iii) sequence analysis of cDNA clones obtained by screening a LuKII library with an IFN-alpha 2 probe. Our data show that more than 90% of the constitutive IFN results from expression of a single gene coding for IFN-alpha 2. The significance of these results is discussed.