This paper describes a sensitive method for analysis of N-linked carbohydrates released enzymatically from within the gel following separation of glycoproteins (50-100 pmols) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated bands containing the glycoproteins were cut from the gel, destained, reduced and alkylated. N-linked glycans were then released by in-gel incubation with peptide N-glycosidase-F (PNGase-F) and extracted with water and acetonitrile. Sialic acid-containing glycans were converted into methyl esters by reaction with methyl iodide, salts and reagents were removed by passage through a mixed-bed column of ion-exchange resins and the glycans were examined by matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. Structural determination of the released glycans was performed by exoglycosidase digestion. Following glycan release and extraction, the protein could be digested within the gel with trypsin, and the masses of the tryptic peptides could be compared with those generated from a sequence database for protein identification. The method is applied to the analysis of N-linked glycans from alpha1-acid glycoprotein from man, cow, sheep and dog. Major species-specific differences in glycosylation were found. Thus, although all four species used N-acetyl-neuraminic acid, only cow and sheep additionally used N-glycolyl-neuraminic acid. Biantennary glycans were the predominant carbohydrates in cow, sheep and dog but man produced more triantennary glycans and a substantial amount of tetraantennary sugars. Fucosylation was only found in glycans from man and cow and both cow and sheep glycans were found to have beta1-3- and well as beta1-4-linked galactose residues in the antennae.