Abstract: The present invention provides an improvement in a method for producing physiologically active proteins by cultivating Escherichia coli having an expression vector which contains a structural gene for such proteins at the downstream end of the translational starting codon. The method comprises cultivating the Escherichia coli in a medium containing (1) an iron ion source, a manganese ion source or a mixture thereof and (2) a nitrogen source derived from natural origin. The advantage of this method is an increase in the yield of physiologically active proteins substantially free of methionine (corresponding to translational starting codon ATG) at the N-terminus.