The amino acid sequence of the porcine pancreatic alpha-amylase chain (496 residues) contains four regions (96-101, 193-201, 233-236 and 295-300) which are highly homologous in amylases of different origins. These regions all belong to the N-terminal domain of the enzyme. Limited proteolysis by subtilisin allows a cut to be made at bond 369-370. Purified fragments indicate that both N- and C-terminal domains are required for amylolytic activity. Kinetic studies and reaction product analysis using oligomaltosides, their nitrophenylated derivatives and amylose as the substrate allowed us to establish: 1) the energy profile of the 5 subsites and, especially, that subsite number 3 is catalytic; 2) that 2 molecules of either maltotriose or its o-nitrophenylated analog or maltose bind to the active site at high substrate concentration. Such a subsite occupancy was confirmed by fluorescence quenching studies. Finally the hydrolysis of p-nitrophenylmaltoside was studied as a function of pH. In contrast to starch hydrolysis, the initial velocity plots for nitrophenol and p-nitrophenylglucoside liberation both gave a narrow pH-activity peak with a maximum value around pH 5.5. All data provide strong evidence for the participation of 2 carboxylic residues in the catalysis.