BACKGROUND: Effect of pH on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH-Mut(+) was investigated at pH = 4.2, 5.0, 5.5, and 6.0. RESULTS: The highest cell concentration was obtained at pH = 6.0 with 53 g L-1, while the highest rhGH concentration was attained at pH = 5.0 as 0.27 g L-1. Total protease secretion increased with increase in pH and with the cultivation time. Oxygen uptake rate increased with increasing pH up to pH = 6.0, having the maximum value, 37 mmol m(-3) s(-1), at pH= 5.5. K(L)a values were similar at all the conditions, having a maximum value of 0.14 s(-1) at pH = 5.0. Taking the final rhGH concentration into account, the most favourable pH was 5.0; where AOX1 expression level showed a similar trend to AOX activity profiles, having the highest value of 9.4 x 10(10) copy mg(-1) CDW at t = 15 h; in parallel to AOX1 expression profile, hGH expression level increased until t = 15 h, with the highest value of 4.0 x 10(10) copy mg(-1) CDW, where a sharp increase in rhGH concentration was obtained. The expression levels of pep4, prb1 and prc1 genes, responsible from the production of proteinase A, proteinase B and, carboxypeptidase Y, were parallel to each other. CONCLUSION: Since it was shown that pH is a crucial operating parameter in fermentation processes using P. pastoris, keeping pH constant at its determined optimum value, pH = 5.0, during the bioprocess is vital in terms of recombinant protein production. (C) 2010 Society of Chemical Industry