Human interferons of lymphoid and nonlymphoid origins were segregated into several fractions by virtue of their elution properties from Blue Sepharose. According to desorption of the main portion of antiviral activity, three major classes could be discerned: 1. Leukocyte interferons eluting predominantly in 0.5 M NaCl buffer, 2. Lymphoblastoid interferons eluting mostly in 1 M NaCl buffer, and 3. Cell culture-derived fibroblast and bladder carcinoma interferons requiring ethylene glycol in addition to 1 M NaCl for elution. Desorption behavior from Blue Sepharose did not necessarily correlate with the presence of specific antigenic markers and no consistent segregation of Le and F antigens in individual fractions was observed. All fractions exhibited comparable activity in heterologous sheep and homologous human cells. Therefore, no distinctive biological features could be associated with multiple interferon species isolated by Blue Sepharose chromatography.