The ultimate activity of an enzyme depends on many regulatory steps from transcription of the gene up to complex formation of the enzyme. Therefore, gene expression (mRNA levels) or protein expression (protein levels) are not reliable parameters to predict the functional activity of an enzyme. Activity measurements in cell homogenates or in frozen or fixed (and thus dead) cell preparations are not appropriate either because post-translational regulation mechanisms that exist in living cells may be lost by homogenization or freezing or chemical fixation of cells. Therefore, metabolic mapping in living cells or, in other words, visualization and quantification using microscopy and image analysis of enzyme reactions in living cells is the approach of choice to understand the functional role of enzymes in vivo as is demonstrated here with a number of examples in recent literature.