UDP-GlcNAc:alpha3-D-mannoside beta1,2- N -acetylglucosaminyltransferase I (GnTI; EC 2.4.1.101) is a Golgi-resident glycosyltransferase that is essential for the processing of oligomannose to hybrid and complex N-glycans in higher eukaryotes. The cDNA of Nicotiana tabacum GnTI has been cloned and characterised previously. To assess the influence of GnTI expression levels on the formation of complex N-glycans we used posttranscriptional gene silencing to knock down the expression of GnTI in the tobacco related species Nicotiana benthamiana. 143 independent transgenic plants containing GnTI constructs in either sense or antisense orientation were generated. 23 lines were selected for measurement of GnTI activity and 10 lines thereof showed a reduction of more than 85% in in vitro assays as compared to wildtype plants. GnTI reduction was stably inherited and did not interfere with the viability of the transformants. Noteworthy one line, 34S/2, exhibited a residual GnTI activity below the detection limit. beta1,2- N -acetylglucosaminyltransferase II (GnTII), an enzyme which acts further downstream in the N-glycosylation pathway, as well as other control enzymes (alpha-mannosidase, beta- N -acetylglucosaminidase) were not affected indicating the specific downregulation of GnTI. Remarkably, immunoblots and mass spectrometric N-glycan profiling revealed no significant changes of the total N-glycan comparable to wildtype plants.