The S1′ substrate specificity of the sunflower seed major aminopeptidase was studied with a series of dipeptide substrates with phenylalanine at P1 and a hydrophobic amino acid at P1′ position. The kinetic parameters of hydrolysis are significantly affected by the structure, side chain hydrophobicity and configuration of the P1′ moiety. Its binding during enzyme-substrate complex formation takes place at a hydrophobic site of limited size following an extraction mechanism as seen from the applied structure-activity correlation. Attempts to establish such dependencies for the catalytic step of the reaction reveal the presence of additional S1′-P1′ enzyme-substrate interactions of greater complexity.