We have previously reported that interleukin (IL)-6 secreted by human fibroblasts induced with either IL-1 or tumor necrosis factor (TNF) consists of at least six differentially modified phosphoglycoproteins of molecular mass 23-30 kDa: a triplet in the mass range from 23 to 25 kDa and another triplet in the range from 28 to 30 kDa. We now report that a combination of metabolic labeling, glycosidase digestion, and lectin chromatography experiments demonstrates that the 23- to 25-kDa species are O-glycosylated and that the 28- to 30-kDa species are both O- and N-glycosylated. Pulse-chase experiments reveal that newly synthesized IL-6 polypeptides rapidly enter two separate protein modification pathways: one leads to O-glycosylation and the other to both N- and O-glycosylation; polypeptides in both pathways are further modified (phosphorylation) prior to secretion. Although both pathways appear to be equally utilized in IL-1- or TNF-induced fibroblasts, the relative proportion of polypeptides proceeding through one or the other pathway can be experimentally modified. In the presence of tunicamycin, IL-6 is secreted exclusively in the O-glycosylated form, whereas in the presence of cycloheximide the pathway leading to both N- and O-glycosylation is dominant. The inclusion of monensin (1 microM) does not inhibit IL-6 secretion from fibroblasts even though it inhibits glycosylation. Combined immunoprecipitation, immunoblotting, and immunoaffinity chromatography experiments reveal additional IL-6 species with mobilities in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions corresponding to molecular masses 17-19 kDa and 45 kDa, suggesting that this cytokine undergoes further alterations. These observations highlight an aspect of IL-6 biosynthesis that appears to represent an excellent model system for studying the mechanisms regulating post-translational protein modifications in human cells and also suggest a basis for reconciling conflicting descriptions of IL-6 structure.