In this work we describe spectroscopic properties of subtilisin DY selectively labeled at alanine 1 by fluorescein isothiocyanate. The reaction takes place in dark at pH 9.3, 20°C, within a 4 h period, in the presence of 5-fold molar excess of the reagent over the protein. As a result, 1.0 mol of fluorescein mol - 1 was covalently bound to the α-amino group of subtilisin DY. Circular dichroism studies showed that the modification created some conformational changes of the proteinase polypeptide chain and the proteolytic activity decreased up to nearly 70% of the control. FTC-subtilisin DY retained a highly ordered structure with 82% of the α-helix structure of the native enzyme. Singlet-singlet energy transfer between the tyrosyl residues of subtilisin DY (donors) and the covalently bound dye (acceptor) was observed. Fluorescence measurements demonstrated that 30% of the light absorbed by the phenolic groups is transfered to the fluorescein group after excitation at 280 nm. An average distance of 28.7±2 9 between the emitting tyrosyl side chains and the bound fluorescein was calculated from energy transfer data. The labeled proteinase can be detected at concentrations as low as 10 - 7 M.