The carbohydrate content and composition of hemocyanins (Hcs) of three prosobranchs (gastropods), Rapana thomasiana, Megathura crenulata and Haliotis tuberculata, were compared. The analyses were performed by gas-liquid chromatography after methanolysis, re-N-acetylation and trimethylsilylation. The two structural subunits of R. thomasiana He, RtH1 and RtH2, both showed 2.6% (w/w) carbohydrate content with very similar monosaccharide composition, indicative for N-glycosylation. The two isoforms of M. crenulata He (KLH), KLH1 and KLH2, on the other hand, definitely differed in glycosylation: KLH2 (3.4% carbohydrate, w/w) comprised relatively less mannose and more N-acetylgalactosamine than KLH1 (3.0% carbohydrate, w/w), in agreement with the fact that O-glycosylation has been observed in a functional unit (FU) of KLH2. For the He of the abalone H. tuberculata, with 4.5% (w/w) carbohydrate, appreciable amounts of 3-O-methyl-D-mannose and 3-O-methyl-D-galactose were detected, showing that the occurrence of methylated sugars is not restricted to the Hcs of pulmonates. From the structural subunit RtH2 of Rapana He the FUs RtH2-b and RtH2-d were isolated. On the basis of amino acid sequence analysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of the respective native and PNGase-F-treated glycopeptides, one N-glycosylation site was found for each FU. This site was located at Asn-405 for RtH2-b and at Asn-394 for RtH2-d; the carbohydrate moiety corresponded to GlcNAc(2)Man(6) and GlcNAc(2)Man(5), respectively. A comparison was made with the N-glycosylation sites of other FUs of Rapana He. (C) 2004 Elsevier Inc. All rights reserved.