Enzyme histochemistry serves as a link between biochemistry and morphology. It is based on metabolization of a substrate provided to a tissue enzyme in its orthotopic localization. Visualization is accomplished with an insoluble dye product. It is a sensitive dynamic technique that mirrors even early metabolic imbalance of a pathological tissue lesion, combined with the advantage of histotopographic enzyme localization. With the advent of immunohistochemistry and DNA-oriented molecular pathology techniques, the potential of enzyme histochemistry currently tends to be underrecognized. This review aims to draw attention to the broad range of applications of this simple, rapid and inexpensive method. Alkaline phosphatase represents tissue barrier functions in brain capillaries, duodenal enterocyte and proximal kidney tubule brush borders. Decrease in enzyme histochemical alkaline phosphatase activity indicates serious functional impairment. Enzyme histochemical increase in lysosomal acid phosphatase activity is an early marker of ischemic tissue lesions. Over the last four decades, acetylcholinesterase enzyme histochemistry has proven to be the gold standard for the diagnosis of Hirschsprung disease and is one of the most commonly applied enzyme histochemical methods today. Chloroacetate esterase and tartrate-resistant phosphatase are both resistant to formalin fixation, EDTA decalcification and paraffin embedding. Early enzyme histochemical insight into development of a pathologic tissue lesion and evaluation of function and vitality of tissue enhance our understanding of the pathophysiology of diseases. In this process, enzyme histochemistry constitutes a valuable complement to conventional histology, immunohistochemistry and molecular pathology for both diagnostic and experimental pathology.