Escherichia coli is one of the most widely used hosts for the production of recombinant proteins. Extracellular protein secretion has the advantage of reducing protein aggregation and simplifying downstream purification. The introduction of five rare codons in a specific region of the alpha-hemolysin (hlyA) gene previously was shown to result in eightfold improvement in secretion of HlyA via the hemolysin (Type-I) pathway. Here we investigate the biological basis for the observed phenomenon that translation rate of HlyA protein may be related to the ability to secrete higher levels of HlyA via the Type-I pathway. A detailed comparative analysis between a hypersecreter mutant strain (hly-slow) and a control strain (hly-parent) shows a significant decrease (by similar to 50%) in the intracellular level of HlyA protein in the hly-slow strain relative to the hly-parent strain. Nearly 100% of the intracellular HlyA protein exists in the inclusion body fraction in both the strains. These results demonstrate the importance of synonymous codon changes in the context of improving HlyA secretion yield via Type-I pathway and further illustrate that production of high levels of secreted proteins appears to require a balance between translation and secretion rate. Biotechnol.