The processing of amino-terminal methionine from cytosolic proteins in E. coli has been investigated in vivo, using amino-terminal-extended human growth hormone (hGH) as a model system. Twenty different hGH-genes with the sequence Met-Xxx-Glu-Glu-hGH where Xxx denotes each of the 20 different amino acids, were constructed and expressed in E. coli. Following purification of the products, the N-terminal amino acid sequences (10 cycles) were determined. The results demonstrate that the removal of methionine is dependent on the amino acid adjacent to methionine, and that the processing is strongly correlated to the radius of gyration of this amino acid. In addition, measurement of the hGH expression level from the 20 clones demonstrated that the small difference in the amino acid extension leads to a change in the specific hGH expression rate.