The kinetics of the α(1–23) peptide, which is the first anti-bacterial peptide to be isolated from a haemoglobin hydrolysate, was studied in the course of peptic hydrolysis at pH 4.5 and 23 °C in an homogenous-phase system. A one-step reversed-phase HPLC coupled with photodiode array detector method was applied to identify and isolate this anti-bacterial peptide. The kinetics of peptide appearance were investigated in acetate buffer alone and in urea as a haemoglobin-denaturing agent. Two different mechanisms, ‘one-by-one’ for native haemoglobin hydrolysis and ‘zipper’ for denatured haemoglobin hydrolysis, were observed. Whatever the haemoglobin state, native or denatured, and whatever the hydrolytic mechanism, one-by-one or zipper, the anti-bacterial α(1–23) peptide is a transient peptide. To prepare the α(1–23) peptide it is suitable to hydrolyse haemoglobin in the presence of urea at a corrected degree of hydrolysis (DHc) of 13.5%. The amount of peptide produced in the presence of urea was twice as high as for the hydrolysis of native haemoglobin. The yields of α(1–23) peptide with respect to haemoglobin at the optimal DHc values were 55 and 25% respectively.