The tyrosyl and tryptophyl fluorescence of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively, is quenched efficiently by I- but is not significantly affected by Cs+. The I-quenching data were analyzed using a modified Stern-Volmer treatment (Lehrer, S.S. (1971), Biochemistry 10, 3254), yielding values for the effective fraction of accessible protein fluorescence of 90-95 and 88-92% for the tyrosyl and tryptophyl emission of diisopropyl-phosphorylsubtilising Carlsberg and Novo, respectively. Similar values were obtained pH 5 and 7. The effective collisional quenching constant depends on pH in a manner suggesting the participation of protein surface charge in the quenching mechanism. Significant singlet energy transfer (efficiency = 0.52) from tyrosyl to tryptophyl residues was inferred from the excitation spectra of diisopropylphosphorylsubtilisn Novo. The very low efficiency of energy transfer to Trp-113 in diisopropylphorphorylsubtilisin Carlsberg suggests that Trp-105 and Trp-241 are the acceptors of tyrosyl emission in the homologous Novo enzyme. The unusually low quantum yield of Trp-113 in diisopropylphosphorylsubtilisin Carlsberg together with the tryptophyl fluorescence quenching behavior of the Novo enzyme suggests that this residue is "buried" and in accessible to quenching in both enzymes. The tyrosyl quenching behavior of diisopropylphorphorylsubtilisin Carlsberg is consistent with the high degree of solvent exposure of aromatic residues evident in the X-ray model of subtilisin Novo.