Gametophytic self-incompatibility, a mechanism that prevents inbreeding in some families of flowering plants, is mediated by the products of a single genetic locus, the S-locus. The products of the S-gene in the female sexual tissues of Nicotiana alata are an allelic series of glycoproteins with RNase activity. In this study, we report on the microheterogeneity of N-linked glycosylation at the four potential N-glycosylation sites of the S2-glycoprotein. The S-glycoproteins from N.alata contain from one to five potential N-glycosylation sites based on the consensus sequence Asn-Xaa-Ser/Thr. The S2-glycoprotein contains four potential N-glycosylation sites at Asn27, Asn37, Asn38 and Asn 150, designated sites I, II, IV and V, respectively. Site III is absent from the S2-glycoprotein. Analysis of glycopeptides generated from the S2-glycoprotein by trypsin and chymotrypsin digestions revealed the types of glycans and the degree of microheterogeneity present at each site. Sites I (Asn27) and IV (Asn138) display microheterogeneity, site II (Asn37) contains only a single type of N-glycan, and site V (Asn150) is not glycosylated. The microheterogeneity observed at site I on the S2-glycoprotein is the same as that observed at the only site, site I, on the S1-glycoprotein (Woodward et al., Glycobiology, 2, 241-250, 1992). Since the N-glycosylation consensus sequence at site I is conserved in all S-glycoproteins from other species of self-incompatible solanaceous plants, glycosylation at this site may be important to their function. No other post-translational modifications (e.g. O-glycosylation, phosphorylation) were detected on the S2-glycoprotein.