The biosynthetic incorporation of tryptophan analogues into proteins using plasmid expression systems has been shown to offer advantages in fluorescence spectroscopic studies of the molecular details of the interacting segments of protein-protein complexes. There are two underlying concepts of this approach. The first is based on a close structural similarity between the analogue and the native tryptophan. The second is that the analogue has an extended red absorbance permitting the selective excitation in the presence of tryptophan. The reported intense absorbance band of 6-azatryptophan at 325 nm would appear to offer a unique opportunity of using this analogue in such studies. In this work, we present the photophysical characterization of the excited state behavior of 6-azatryptophan through a complete study of the cognate indole moiety, 6-azaindole. It was found that the absorbance at 320 nm for 6-azaindole and 325 nm for 6-azatryptophan was due to the protonated form in which both ring nitrogens are protonated. The protonation equilibrium for 6-azaindole had a pK(a) value of 8. The fluorescence of 6-azaindole was centered at 380 nm. It was found that the neutral form of 6-azaindole was nonfluorescent in nonpolar solvents. In aqueous solution at pH values between 6.5 and 10, the fluorescence maximum of 6-azaindole remained at 380 nm, and the intensity decreased as the pH increased. Above pH 10, there was increased fluorescence on the low-energy side of the spectrum until at pH 14 the fluorescence spectrum showed a single maximum at 440 nm. The observations of the collective experiments in a variety of solvent conditions and pH values, including time-resolved fluorescence measurements, permitted a consistent assignment of each of the fluorescence species together with a rationalization of the excited-state processes that result from the excitation of either the protonated form or the neutral NI-H tautomer. The 440 nm fluorescence was attributed to the fluorescence from the excited state of the N6-H tautomer, a species that formed from the protonated excited state molecule that had previously been formed from initial excitation of the NI-H ground state. The work reports determinations of the excited-state pK(a) values of each of the three species. The relative energy values of each of the ground-state molecules and excited-state species could be estimated. The relevance of the photophysics of 6-azaindole to protein structural studies was demonstrated by the biosynthetic incorporation of 6-azatryptophan into the Y99W mutant of rat calmodulin and the observation of 6-azaindole fluorescence from this protein.