The effect of water on the reaction of 3-aminopropyltriethoxysilane (APTES) with mesoporous silica is investigated on two model systems. A MCM-41 type material with a well-defined pore size is used to investigate changes in the pore size distribution upon reaction with APTES in toluene containing various amounts of water. It is found that with increasing amount of water, clustering of APTES occurs, leading to a non-uniform distribution of the grafted amino groups and a scarcely functionalized pore body. A second model system with defined particle morphology and one-dimensional channels is employed to visualize the distribution of the grafted amino groups by fluorescent labeling and confocal laser scanning microscopy. The combination of nitrogen sorption and confocal laser scanning microscopy provides valuable insights concerning the role of trace water in the functionalization of mesoporous silica with alkoxysilanes.