The fluorescence properties of proteinase K are described and related to the X-ray model refined at 1.48 A resolution. Upon excitation of proteinase K at 295 nm the fluorescence is determined by the two tryptophan residues, Trp-8 and Trp-212. The tryptophans are partly buried just below the surface of the molecule. Neither Trp is in a highly hydrophobic environment, suggesting that this cannot be the explanation for the fluorescence at 330 nm: formation of exiplexes with adjacent peptide bonds would seem to be the more likely cause. Trp-8 is located in a 'cavity', close to an internal cluster of water molecules. The contribution of Trp-8 to the total indole emission is 60% and that of Trp-212 is 40%. The tryptophan fluorescence quantum yield is constant in the pH range 3-9. The fluorescence spectrum resulting from the simultaneous excitation of the tyrosyl and tryptophyl residues at 280 nm is dominated by the indole fluorophores: 61% of the light absorbed by the tyrosyl side chains is transferred to the two indole rings. Iodide and caesium are not efficient quenchers of the proteinase K tryptophan fluorescence, which is explained by restricted access of the ions to the somewhat buried Trp side chains and by electrostatic repulsion of caesium ions. Acrylamide quenching proceeds via both a dynamic and a static process and the data show homogeneity of the indole fluorescence arising from fluorophores in similar environments. The activation energy for the thermal deactivation of the excited tryptophans is 54 kJ mol-1. This value is substantially higher than those found for other proteinases from microorganisms and arises from the thermostability of proteinase K. Photooxidation of proteinase K in the presence of proflavine follows the kinetics of a first order reaction. The two tryptophans differ in their photoreactivity, Trp-212 being considerably more reactive.