The yeast Pichia pastoris is a convenient production system that enables expression of heterologous proteins in high amounts. As a fermentation method, shaking flasks are very popular because of their simplicity of handling and their low cost. We compared the expression level of the enzyme acetylcholinesterase in a transformed strain of P. pastoris grown in different flasks, presenting various designs but all with the same volume. A several-thousand-fold difference appeared in the expression levels; and the results could not be explained by differences between the flasks in the oxygenation of the medium. The data show that flask design is an important factor to consider for optimising fermentation processes.