The extracellular domain of human EGF receptor (sEGFR) produced by CHO cells has been used in various biophysical studies to elucidate the molecular mechanism of EGFinduced receptor activation. We have found that the CHO sEGFR contains one oligosaccharide chain attached to an atypical N-glycosylation consensus sequence, Asn³²-X³³-Cys³⁴. The oligosaccharide structure at Asn³² is a mixture of the monosialo and asialo forms of a core fucosylated biantennary complex-type oligosaccharide. Deletion of this atypical glycosylation site by replacement of Asn³² with lysine changed neither the expression nor function of the full length EGFR in CHO cells. The glycosylation at Asn³² in CHO sEGFR was incomplete: 20% of Asn³² remained unmodified. Thus, CHO sEGFR itself is heterogeneous with respect to the glycosylation at Asn³², which may cause problems in biophysical studies. An attempt to remove the oligosaccharide at Asn³² enzymatically did not succeed under nondenaturing conditions. Therefore, sEGFR with the mutation of Asn³²→Lys³² is useful for biophysical and biochemical studies, and, particularly, for X-ray crystallography.