To establish general rules for setting up an enzyme microreactor system, we studied the effect of diffusion on enzyme activity in a microreactor. As a model system we used the hydrolysis of ortho-nitrophenyl-beta-D-galactopyranoside by beta-galactosidase from Kluyveromyces lactis. We found that the Michaelis-Menten kinetic parameters were similar at the microscale and bench scale. With residence times below a few seconds, diffusion effects limited the reaction rate and therefore reduced the conversion per volume of enzyme microreactor. The critical residence time where diffusion limits the conversion increased quadratically with channel width, increased with enzyme concentration, and decreased with substrate concentration. These general rules can be used for choosing parameters when setting up an enzyme microreactor system. To use an enzyme microreactor efficiently, diffusion effects should be taken into account. (C) 2010 Elsevier B.V. All rights reserved.