The biosynthesis of human CD8 glycoprotein in transfected rat epithelial cells produces an unglycosylated precursor, an intermediate species only initially O-glycosylated, and a doublet mature form carrying neutral and sialylated O-linked oligosaccharides with the core-2 structure (Pascale, M. C., Malagolini, N., Serafini-Cessi, F., Migliaccio, G., Leone, A., and Bonatti, S. (1992) J. Biol. Chem. 267, 9940-9947). In this study the most relevant post-translational events: dimerization, addition of the first O-linked GalNAc, fulfillment of O-linked chains, as well as expression of involved glycosyltransferases, have been examined and correlated with the localization and transit rate of CD8 through the exocytic pathway. The glycosyltransferase activities measured in rat epithelial cells transfected with human CD8 DNA are entirely consistent with the primary structure assigned to CD8 oligosaccharides. The half-time of appearance of the initially O-glycosylated precursor and mature form was estimated to be 4 and 14 min, respectively, and the half-time for delivery of mature CD8 to the cell surface was found to be about 30 min, indicating a very fast routing. Pulse experiments with [35S]cysteine at 37 degrees C followed by chase-periods at low temperatures showed that folding/dimerization occurs before routing to the Golgi apparatus, whereas the addiction of O-linked GalNAc appears to take place later, very likely in cis-Golgi cisternae. Treatment of cells with monensin accumulated the intermediate CD8 form carrying non-elongated O-linked GalNAc, whereas brefeldin A treatment produced a sialylated glycoprotein species with a mobility faster than the mature CD8. These results indicate that the two drugs affect assembly of O-linked chains at different time of their processing.