Human fibroblast interferon binds to a concanavalin A-agarose (Con A-Sepharose) equilibrated with methyl alpha-D-mannopyranoside, or levan; in contrast, it is only partially retarded on a similar column equilibrated with ethylene glycol. Interferon does not bind, however, to a lectin column equilibrated with both methyl alpha-D-mannopyranoside and ethylene glycol. Thus, a hydrophobic interaction between fibroblast interferon and the immobilized lectin seems to account for a large portion of the binding forces involved. Other hydrophobic solutes, such as dioxane, 1, 2-propanediol, and tetraethylammonium chloride, were found equally or more efficient than ethylene glycol in displacing interferon from the lectin column. The elution pattern of interferon from a concanavalin A-agarose (Con A-Sepharose) column, at a constant ehtylene glycol concentration and with an increasing mannoside concentration, reveals the existence of four distinct interferon components. The selective adsorption to and elution from a concanavalin A-agarose (Con A-Sepharose) column resulted in about a 3000-fold purification of human fibroblast interferon and complete recovery of activity. The specific activity of the partially purified interferon preparation is about 5 X 10(7) units per mg of protein. The chromatographic behavior of human leukocyte interferon is remarkable in that it does not bind to concanavalin A-agarose at all indicating the absence of carbohydrate moieties recognizable by the lectin, or if present, their masked status. When concanavalin A was coupled to an agarose matrix (cyanogen bromide activated) at pH 8.0 and 6.0 human fibroblast interferon bound to both lectin-agarose adsorbents and could be recovered with methyl alpha-D-mannopyranoside. Concanavalin A, immobilized directly on agarose matrix at pH 8.0 and 6.0, thus displays only carbohydrate recognition toward interferon. By contrast, unless a hydrophobic solute was included in the solvent containing methyl mannoside, human fibroblast interferon could not be recovered from concanavalin A-agarose coupled at pH 9.0. When concanavalin A was immobilized via molecular arms, in tetrameric as well as dimeric forms, the binding of interferon again occurred exclusively through carbohydrate recognition. Thus, the hydrophobic interaction can be eliminated by appropriate immobilization of the lectin, and then adsorbed glycoproteins, as exemplified here by interferon, can be recovered readily with methyl mannoside alone.