Enzyme kinetics studies have been usually designed as dilute solution experiments, which differ substantially from in vivo conditions. However, cell cytosol is crowded with a high concentration of molecules having different shapes and sizes. The consequences of such crowding in enzymatic reactions remain unclear. The aim of the present study is to understand the effect of macromolecular crowding produced by dextran of different sizes and at diverse concentrations in the well-known reaction of oxidation of NADH by pyruvate catalyzed by L-lactate dehydrogenase (LDH). Our results indicate that the reaction rate is determined by both the occupied volume and the relative size of dextran obstacles with respect to the enzyme present in the reaction. Moreover, we analyzed the influence of macromolecular crowding on the Michaelis-Menten constants, v(max) and K-m. The obtained results show that only high concentrations and large sizes of dextran reduce both constants suggesting a mixed activation diffusion control of this enzymatic reaction due to the dextran crowding action. From our knowledge, this is the first experimental study that depicts mixed activation diffusion control in an enzymatic reaction due to the effect of crowding.