Cytosolic and nuclear O-linked N-acetylglucosaminylation has been proposed to be involved in the nuclear transport of cytosolic proteins. We have isolated nuclear and cytosolic N-acetyl-d-glucosamine (GlcNAc)-specific lectins from adult rat liver by affinity chromatography on immobilized GlcNAc and identified these lectins, by a proteomic approach, as belonging to the heat-shock protein (HSP)-70 family (one of them being heat-shock cognate 70 stress protein). Two-dimensional electrophoresis indicated that the HSP-70 fraction contained three equally abundant proteins with molecular masses of 70, 65 and 55 kDa. The p70 and p65 proteins are phosphorylated and are themselves O-linked GlcNAc (O-GlcNAc)-modified. The HSP-70 associated into high molecular mass complexes that dissociated in the presence of reductive and chaotropic agents. The lectin(s) present in this complex was (were) able to recognize cytosolic and nuclear ligands, which have been isolated using wheat germ agglutinin affinity chromatography. These ligands are O-GlcNAc glycosylated as demonstrated by [(3)H]galactose incorporation and analysis of the products released by reductive beta-elimination. The isolated lectins specifically recognized ligands present in both the cytosol and the nucleus of human resting lymphocytes. These results demonstrated the existence of endogenous GlcNAc-specific lectins, identified as HSP-70 proteins, which could act as a shuttle for the nucleo-cytoplasmic transport of O-GlcNAc glycoproteins between the cytosol and the nucleus.