The covalent structure of bovine brain calreticulin, a major Ca(2+)-binding protein in the lumen of the endoplasmic reticulum, was determined by analysis of the purified protein. The protein consisted of 400 amino acids, with an N-linked oligosaccharide attached to the polypeptide chain. The polypeptide sequence determined was compatible with the sequence of calreticulin deduced from cDNA of different sources, with a number of differences presumably due to species-specific amino acid substitutions. The protein retained the C-terminal tetrapeptide, KDEL, involved in retention of proteins resident in the endoplasmic reticulum, whereas the N-terminal signal peptide predicted from the cDNA sequence had been removed in the purified protein. The bovine brain protein contained a high-mannose type of oligosaccharide attached to Asn162, which is typical of resident endoplasmic reticulum proteins. The carbohydrate moiety was heterogeneous and had the composition GlcNAc2Man4-9, of which GlcNAc2Man5 was the most abundant in the bovine brain preparation. Glycosylation of calreticulin, however, appeared to be a species-specific modification, as Asn162 is replaced by Asp in the sequences already determined for a number of species. Analysis of the purified protein also identified an intramolecular disulphide bridge between Cys120 and Cys146.