The high stability and productivity of recently-developed Escherichia coli JM101 strains expressing human epidermal growth factor (hEGF) facilitated scale-up of hEGF production, and a protocol to purify hEGF from bacterial culture supernatant was developed. hEGF-containing supernatant from an induced hEGF-expressing recombinant E. coli culture was purified by: (A) QAE Sephadex A-25 ion-exchange chromatography; (B) Sephadex G-25 desalting; (C) SP-Sepharose cation-exchange chromatography; and (D) reverse-phase HPLC. The hEGF obtained was pure by HPLC and SDS-PAGE. The N-terminus of the purified hEGF was authentic. Commercial pure hEGF, and hEGF purified as described, were assessed for bioactivity, and yielded superimposable curves. The recovery of hEGF with this protocol was 30% of original, while the purity was 97-100%.