Microchannel reactor technologies are gaining widespread use in a large range of areas, which comprise biotechnology and chemistry. The small volumes involved and the favorable mass and heat transfer inherent to these devices make them particularly useful for the screening of biocatalysts and rapid characterization of bioconversion systems. In the present work, the enzymatic oxidation of cholesterol to 4-cholesten-3-one performed within microchannels by cholesterol oxidase, was studied in a two-phase system, comprising an organic phase as substrate and product pool and an aqueous phase with dissolved enzyme. A mathematical model based on mass balances for cholesterol, 4-cholesten-3-one and dissolved oxygen concentrations, comprising double-substrate Michaelis-Menten kinetics and the velocity profile of two immiscible fluids, was developed in order to describe and predict the process of cholesterol oxidation. The numerical procedure of solving the non-linear 3D model was based on an implicit finite-difference method improved by non-equidistant differences. In a Y-shape microreactor geometry, roughly up to 70% conversion of cholesterol was achieved at residence times below 1 min. The suitable adjustment of the ratio of the fluid flow rates was performed by taking into account the viscosity of the fluids involved. This allowed for phase separation to be reestablished at the Y-shaped exit from the microreactor and thereby enabled in situ product separation from the aqueous phase containing the enzyme. (c) 2010 Elsevier B.V. All rights reserved.