The fluorescence of lima bean trypsin inhibitor is due to a single tyrosine residue at position 69. The lifetime of this tyrosine fluorescence is 620 +/- 50 ps (mean +/- SD) and is little affected by addition of 0.88 M citrate, an efficient quencher of tyrosine fluorescence. The steady-state emission intensity is also only weakly reduced by the quencher. The tyrosine is thus not accessible to the citrate and is probably located in the interior of the protein. The high pK of the tyrosine supports this conclusion. The fluorescence anisotropy decay of the inhibitor's tyrosine can be fitted to a double exponential form, with time constants of about 40 ps and greater than or equal to 3 ns. The anisotropy at time zero is 0.19 +/- 0.015 (mean +/- SD), the same as for N-acetyl-L-tyrosinamide in viscous glycerol solution. The nanosecond component of the decay is consistent with rotation of the entire protein molecule. The 40-ps component demonstrates that the tyrosine has considerable freedom to move independently of the protein as a whole. This rotational correlation time is approximately what is observed for free tyrosine in aqueous solution. Since the polypeptide chain near tyrosine-69 is anchored by several disulfide bonds, the data argue that this interior portion of the protein consists of a rigid, immobile backbone embedded in fluid, mobile amino acid side chains.