We have biosynthetically incorporated several tryptophan analogues into three forms of Staphylococcal nuclease to investigate the spectroscopic characteristics of these "intrinsic" probes and their effect on the structure of the proteins. The set of tryptophan analogues includes 5-hydroxytryptophan, 7-azatryptophan, 4-fluorotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan. 5-Hydroxytryptophan and 7-azatryptophan have red-shifted absorbance spectra, and the latter has a red-shifted fluorescence, which is very sensitive to its environment (being heavily quenched in water). The fluorotryptophans can serve as 19F NMR probes, and 4-fluorotryptophan has a very low fluorescence quantum yield, thus making it a "knock-out" fluorescence analogue. The set of proteins studied includes wild-type nuclease, which has a single tryptophan site at position 140; its V66W mutant, which has a second tryptophan at position 66; and the Delta 137-149 fragment, V66W', which only has a tryptophan at position 66. The environments of positions 66 and 140 are significantly different; position 140 is near the end of the long C-terminal alpha-helix and is moderately solvent-exposed, whereas position 66 is in the beta-barrel core region of the protein and is surrounded by apolar side chains. Absorbance and 19F NMR spectra are used to estimate the extent of analogue incorporation for each protein. Steady-state and time-resolved fluorescence data are reported to characterize the emission of the analogues in these positions in the three proteins and to develop the use of the analogues as probes of protein structure and dynamics. Circular dichroism spectra are reported to show that, in all but a couple of cases, the secondary structure of the proteins containing the analogues is not significantly perturbed by the probes. Additionally, fluorescence anisotropy decay data show the variants of wild-type nuclease to have a rotational correlation time similar to that of tryptophan-containing nuclease.