Total amylase activity in serum and urine is formed by pancreatic (P) and salivary (S) isoenzymes. The evaluation of isoamylases provides better information on enzyme changes during the disease than total activities alone. The resolution of pancreatic from extrapancreatic origin of hyperamylasemia may be clinically important. The experience obtained from the analysis of isoamylases in more than 1500 patients with different clinical diagnoses we compare with a contemporary knowledge of disturbances in amylase activities. We developed a method separating quantitatively both isoamylases on the mini-columns of ion-exchanger which we used in routine clinical investigation. In the first section we selected the findings on physiology and biochemistry of isoamylases. We described for the first time a significant decrease of P-isoamylase activity in serum during the intravenous infusions of hypertonic glucose, amino acids and during acute hypercalcaemia. We suggested that hypertonic glucose, amino acids and calcium may regulate directly or indirectly the amylase flux from acinar cells in the pancreas across basolateral membrane into blood. This endocrine secretion of amylase may be important in different clinical conditions in which changes of neurohumoral and/or hormonal regulation are developed. The isoamylase activities in patients with different diagnosis are analyzed in the clinical section. The results may be correctly evaluated only in connection with the pathogenesis of isoamylase changes. Disorders of the organs producing amylase (i.e. pancreas or salivary glands) may induce changes of isoamylases depending on their functional status. A progressive loss of amylase producing cells may be accompanied by a decrease of enzyme activity in serum as was described in chronic pancreatitis with exocrine insufficiency. However, the amylase activity in serum is significantly influenced by clearance mechanisms, too. Disorders of the liver or kidneys are accompanied predominantly with hyperamylasemia caused by the disturbed clearance mechanisms. The amylase activity in serum is a consequence of the result between input and output of the enzyme within the blood stream. Some humoral and hormonal regulations are able to modulate both processes in vivo. We suppose that pathogenetic standpoint has the main role for correct interpretation of isoamylase activities. The pathogenesis of hyperamylasemia is therefore discussed in single chapters. In conclusion, the isoamylase activities in serum and urine are influenced beside genetic background by many factors in health and disease which may be respected during the evaluation of the results.