Fluorescence of rat serum albumin (RSA) was compared with that of human serum albumin (HSA). Both proteins have a single Trp residue located in a hydrophobic region. Fluorescence excitation and emission spectra of both proteins are identical and are characteristic of the emission of a Trp residue embedded in a hydrophobic region. Each of the two proteins emits with 3 fluorescence lifetimes and identical pre-exponentials. These 3 lifetimes have been measured in a large number of proteins and therefore characterize the intrinsic properties of tryptophan. The pre-exponential values represent the populations of fluorophores that emit. Addition of 6,P-toluidinyl-naphthalene-2-sulfonate (TNS) to RSA or HSA decreases the fluorescence intensity of the Trp residue following the binding of TNS to the protein and results in new fluorescence lifetimes characteristic of TNS bound to the protein.