The filament forming ability and post-translational modifications of the human intermediate filaments, keratin polypeptides 8 and 18 (K8/18), were studied in recombinant baculovirus-infected insect (Spodoptera frugiperda, Sf9) cells. No change in cell morphology was noted after high levels of K8/18 were expressed in Sf9 cells coinfected with recombinant virus-containing human K8 and K18. Immunofluorescence staining showed that K8/18 expressed in Sf9 cells formed somewhat what disorganized and rope-like filaments, in contrast with K8 or K18 expression alone, which did not result in filament formation. K8/18 expressed in Sf9 cells were glycosylated (O-linked N-acetylglucosamine) and phosphorylated, and each modification occurred on different molecules of K8 and K18, as previously found in human HT29 cells. The glycosylation and phosphorylation of K18 in human and insect cells were very similar as determined by tryptic peptide mapping and localization to the head and proximal rod domains. In contrast, differences were noted in the relative intensity of the tryptic phospho- and glycopeptides of K8 expressed in human and insect cells and in the ratio of K8 to K18 phosphorylation in human and insect cells. Our results show that although quantitative differences exist, the post-translational modification of K8/18 expressed in insect cells is quite similar to its mammalian counterpart, especially for K18. Baculovirus expressed K8/18 should prove useful for mapping phosphorylation and glycosylation sites and for studying factors involved in organized filament assembly in mammalian cells.