The hemocyanin of the crab Carcinus aestuarii contains a carbohydrate moiety that represents 1.6% of protein mass. This carbohydrate content is higher than that exhibited by other arthropod hemocyanins so far investigated. By combination of FPLC ion exchange chromatography and reverse-phase HPLC, the native oligomeric protein can be resolved into three major and one minor electrophoretically pure fractions that are found to be homogeneous by N-terminal sequencing and correspond to the subunit polypeptide chains. Sugar analysis on the different subunits reveals that the subunit referred to as Ca2 is glycosylated, with a carbohydrate content of 6.3%. By Ca2 trypsin digestion, separation of glycopeptides, and amino acid sequencing, three consensus sequences for O-glycosylation and one for N-glycosylation were found. MALDI-MS was applied for the determination of the molecular masses of the various glycopeptides and peptides after removal of carbohydrates by neuraminidase and alpha-N-acetylgalactosaminidase.