Gp62 is a nuclear pore complex glycoprotein of vertebrates containing multiple O-linked N-acetylglucosamine monosaccharides. We have recently shown (Cordes et al., Eur. J. Cell Biol. 55, 31-47 (1991)) that gp62 of mouse and Xenopus laevis is predominantly glycosylated in the amino-terminal half of the molecule. Here we describe in vitro glycosylation in rabbit reticulocyte lysate of gp62 and of individual segments of this protein expressed as recombinant polypeptides in E. coli. Competition experiments between purified polypeptides revealed that gp62 exhibits in vitro at least two types of binding sites for a cytosolic N-acetylglucosaminyltransferase resulting in a sequential glycosylation of the protein. High affinity binding sites for this glycosyltransferase were located in the amino-terminal domain between amino acids 248-341 of mouse gp62, whereas low affinity binding sites were scattered in the entire amino-terminal half. The segment containing amino acids 248-341 was able to compete with in vitro O-glycosylation of gp62 subdomains containing low affinity binding sites. This gp62 segment was also able to compete with O-glycosylation of other unrelated cellular proteins in vitro.