Human epidermal growth factor (h-EGF) is 53 amino acid polypeptide that stimulates proliferation and differentiation of various cells. To identify critical residues that govern biological activity of h-EGF, six analogues having specific amino acid substitution at 13, 15, 37 and 41 were synthesized by stepwise solid phase method using the Boc-HF strategy. Homogenity and purity of the synthesized peptides were performed by RP-HPLC, FAB-MS, amino acid analysis and thermolytic digestion. The biological activity of these h-EGF analogues was measured by mitogenic stimulation in NIH/3T3 cells. The substitution of aromatic amino acid (Phe) at position 37 and 13 had little effect on mitogenic activity of h-EGF. In contract, the replacement of Ala at these positions occurred a big loss of acitivity $(5{ imes}10^{-2} ; and ; 10^{-3}-fold)$. This result indicate that aromatic side chain at position 13 and 37 play an important role in biological activity of h-EGF. The semiconservative substitution of Ala for Leu at position 15 and conservative change of Lys for Arg at position 41 also drastically reduced mitogenic activity $(10^{-4} ; and ; 10^{-5}-fold)$. Thus, the hydrophobic bulky group at postion 15 and the guanidino group at position 41 were essential for the biological activity of h-EGF.