The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 ± 1.0 nm (A) and 358.5 ± 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 ± 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70°C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 ± 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra. © 2002 Japanese Biochemical Society.