Human immune interferon, induced in leukocytes by phytohemagglutinin, was prepared in the absence and presence of tunicamycin, an antibiotic which selectively inhibits the glycosylation of newly synthesized glycoproteins. Interferon preparations, produced in the absence of the antibiotic, displayed a considerable chromatographic heterogeneity on: (a) concanavalin A-agarose, (b) phenyl-agarose, (c) Cibacron Blue F3GA-agarose, and (d) polyuridylic acid-agarose. This heterogeneity was completely eliminated when tunicamycin (2 microgram/ml) was present during induction of interferon; all activity was then recovered in the breakthrough fractions from all sorbents. The level of interferon activity in leukocyte culture fluid was not affected by tunicamycin within the range of concentration 0.05 to 2.0 microgram/ml. These data indicate that (a) human immune interferon undergoes glycosylation, and tunicamycin is an effective inhibitor of this process. Thus, it appears that (b) at least some of the carbohydrates of human immune interferon are N-glycosidically linked. Moreover, it seems that (c) glycosylation is not necessary for an interferon molecule to either be secreted by the cell or (d) to express its antiviral function. Such properties of human immune interferon as (e) the apparent hydrophobicity and (f) an affinity for a polyribonucleotide are conferred only when its glycosylation is unimpaired.