A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate‐limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide‐bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H‐sensitivity and located to the endoplasmic reticulum and the cis‐Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D‐glucose and L‐glutamine, on the ATIII‐yield. We found that utilization of D‐glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII‐synthesized per L‐glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L‐glutamine. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 131–140, 2002; DOI 10.1002/bit.10206