High molecular weight human epidermal growth factor (HMW-hEGF) was purified to homogeneity from human urine. The purification was achieved exclusively by the use of immunoaffinity chromatography and reverse phase high performance liquid chromatography (HPLC). The purified HMW-hEGF is composed of a single polypeptide chain with an apparent molecular weight of 30,000 and has pI of 4.0. Its N-terminal sequence was determined as Val-Ser-Asp-Gln-Asp-Asp-( )-Ala-Pro-Val-Gly-( )-Ser-Met-Tyr-Ala-Arg-( )-Ile- Ser-. The trypsin treatment of this protein gave hEGF like fragments. These results suggest that the obtained HMW-hEGF may play an important role in biosynthetic precursor of hEGF.