Recombinant human erythropoietin (EPO) is a glycoprotein produced as a therapeutic agent from mammalian cell cultures for the treatment of anemia associated with severe kidney damage. The EPO structure has a high glycan content which is essential for bioactivity but shows considerable molecular heterogeneity. The cell culture conditions that affect the heterogeneity of the glycoforms of EPO are not well understood. However, the accumulation of ammonia in culture is one factor that has been associated with an enhanced heterogeneity of glycoforms. In this report we investigate the metabolic perturbations associated with ammonia and glucosamine that may give rise to an altered pattern of EPO glycosylation. Recombinant human erythropoietin was synthesized in serum-free cultures of transfected Chinese hamster ovary (CHO) cells. The molecular heterogeneity of erythropoietin was increased by supplementation of cultures with either ammonia or glucosamine. The enhanced molecular heterogeneity was shown to be due to variable glycosylation that resulted in EPO with an enhanced molecular weight and isoelectric point range. Enzymatic removal of the glycan moiety of EPO in all cases resulted in a single molecular form with a molecular weight of 18 000, which corresponded to non-glycosylated EPO. The variable glycosylation was consistent with reduced sialylation and antennarity of the carbohydrate structures present on the three N-linked sites of EPO. In the presence of ammonia (>30 mM) the proportion of tetrasialylated and tetraantennary glycan structures were reduced by 73% and 57%, respectively, as determined by HPLC analysis. Such changes were also observed, although to a lesser extent (41% and 37%), by an increase in the glucosamine concentration (>10 mM) in the medium. The enhanced heterogeneity of the glycan structures coincided with a significant increase in the intracellular UDP− N-acetylhexosamine (UDP−GNAc) pool. The measured UDP−GNAc level was up to 2 orders of magnitude higher in the presence of either glucosamine or ammonia. However, the changes in the glycosylation profiles induced by either glucosamine or ammonia were significantly different even at the same intracellular UDP-GNAc concentration. This suggests that the enhanced EPO heterogeneity could not be mediated solely by the increased UDP−GNAc level. Glucosamine (but not ammonia) was shown to cause significant inhibition of glucose transport into the cells, which could induce a different pattern of primary metabolism.