Our previous study has shown that urinary bikunin has four major isomers, which differ with respect to the sulfation ratio in a glycosaminoglycan chain but which do not exhibit any differences in amino acid composition, N-terminal amino acid sequence, C-terminal amino acid, sialic acid and uronic acid content. In this study, the amino acid sequences of the four isomers were examined. Sequence analysis showed that the amino acid sequence of the four isomers was completely identical and was also the same as the amino acid sequence deducted from cDNA encoding bikunin. We conclude that the charge heterogeneity of urinary bikunin is due to only a difference in the sulfation ratio of a glycosaminoglycan chain.