Peptide chloromethanes with the general formula dansyl-(Ala)n-Phe-CH2Cl where n = 0, 1, 2, 3 and dansyl fluoride were used to investigate the substrate-binding sites A and B in subtilisins DY and Carlsberg. Kinetic evidence for the introduction of the dansyl group at the subsites S2, S3, S4 and S5 were obtained. Fluorescence experiments showed that the micro-environment of these subsites is quite apolar. However, some differences in their accessibility to external reagents can be revealed in fluorescence quenching experiments. Efficient singlet-singlet radiationless energy transfer from the single Trp 113 to the dansyl group selectively bound at the respective subsites was observed and intramolecular distances between the chromophores were determined. The values calculated for the pairs Trp 113 plus Dns at S2, Trp 113 plus Dns at S4 and Trp 113 plus Dns at S5 are practically identical (1.7-2.0 nm) for the two enzymes. Conclusions on the shape of the substrate-binding sites in subtilisins DY and Carlsberg are drawn. The mutual spatial orientation of the donor (Trp 113) and acceptor (Dns at Sn) dipoles is also elucidated.